Heregulin variants

ABSTRACT

The present invention provides heregulin variants that are capable of binding an ErbB receptor. Included in the invention are variants of human heregulins, and, in particular, variants of human heregulin-β1 having enhanced affinity for the ErbB-3 and ErbB-4 receptors. These variants include at least one amino acid substitution and can include further modifications. The invention also provides nucleic acid molecules encoding heregulin variants and related vectors, host cells, pharmaceutical compositions, and methods.

GOVERNMENT LICENSE RIGHTS STATEMENT

The U.S. Government has a paid-up license in this invention and theright in limited circumstances to require the patent owner to licenseothers on reasonable terms, as provided for by the terms of Grant No.GM16549-01 awarded by the National Institutes of Health.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of provisional applicationSer. No. 60/037,581, filed Feb. 10, 1997, which is expresslyincorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to heregulin variants, nucleic acidmolecules encoding such variants, and related vectors, host cells,pharmaceutical compositions, and methods. In particular, the inventionrelates to amino acid substitution variants of human heregulin-β1 havingan enhanced affinity for the ErbB-3 and ErbB-4 receptors.

2. Description of the Related Art

Transduction of signals that regulate cell growth and differentiation isregulated in part by phosphorylation of various cellular proteins.Protein tyrosine kinases are enzymes that catalyze this process.Receptor protein tyrosine kinases are believed to direct cellular growthvia ligand-stimulated tyrosine phosphorylation of intracellularproteins. Growth factor receptor protein tyrosine kinases of the class Isubfamily include the 170 kilodalton (kDa) epidermal growth factorreceptor (EGFR) encoded by the erbB1 gene. erbB1 has been causallyimplicated in human malignancy. In particular, increased expression ofthis gene has been observed in more aggressive carcinomas of the breast,bladder, lung, and stomach.

The second member of the class I subfamily, p185^(neu) (also called theErbB-2 receptor or p185^(HER2)), was originally identified as theproduct of the transforming gene from neuroblastomas of chemicallytreated rats. The neu (erbB2 or HER2) gene encodes a 185 kDa receptorprotein tyrosine kinase.

Amplification and/or overexpression of the human erbB2 gene correlateswith a poor prognosis in breast and ovarian cancers. Slamon et al.,Science 235:177-82 (1987); Slamon et al., Science 244:707-12 (1989).Overexpression of erbB2 has been correlated with other carcinomasincluding carcinomas of the stomach, endometrium, salivary gland, lung,kidney, colon and bladder. Accordingly, in U.S. Pat. No. 4,968,603,Slamon et al. describe and claim various diagnostic assays fordetermining erbB2 gene amplification or expression in tumor cells.Slamon et al. discovered that the presence of multiple copies of theerbB2 oncogene in tumor cells indicates that the disease is more likelyto spread beyond the primary tumor site, and that the disease maytherefore require more aggressive treatment than might otherwise beindicated by other diagnostic factors. Slamon et al. conclude that theerbB2 gene amplification test, together with the determination of lymphnode status, provides greatly improved prognostic utility.

A further related gene, called erbB3 (or HER3), which encodes the ErbB-3receptor (p180^(HER3)) has also been described. See U.S. Pat. No.5,183,884; Kraus et al., PNAS USA 86:9193-97 (1989); EP PatentApplication No. 444,961A1; Kraus et al., PNAS USA 90:2900-04 (1993).Kraus et al. (1989) discovered that markedly elevated levels of erbB3mRNA were present in certain human mammary tumor cell lines indicatingthat erbB3, like erbB1 and erbB2, may play a role in human malignancies.Also, Kraus et al. (1993) showed that EGF-dependent activation of theErbB-3 catalytic domain of a chimeric EGFR/ErbB-3 receptor resulted in aproliferative response in transfected NIH-3T3 cells. Furthermore, theseresearchers demonstrated that some human mammary tumor cell linesdisplay a significant elevation of steady-state ErbB-3 receptor tyrosinephosphorylation, further implicating this receptor in humanmalignancies. The role of erbB3 in cancer has been explored by others,and this gene has been found to be overexpressed in breast (Lemoine etal., Br. J. Cancer 66:1116-21 [1992]), gastrointestinal (Poller et al.,J. Pathol. 168:275-80 [1992]; Rajkumer et al., J. Pathol. 170:271-78[1993]; Sanidas et al., Int. J. Cancer 54:935-40 [1993]), and pancreaticcancers (Lemoine et al., J. Pathol. 168:269-73 [1992], and Friess etal., Clinical Cancer Research 1:1413-20 [1995]).

The class I subfamily of growth factor receptor protein tyrosine kinaseshas been further extended to include the ErbB-4 (HER4) receptor, whichis the product of the erbB4 (HER4) gene. See EP Patent Application No.599,274; Plowman et al., PNAS USA 90:1746-50 (1993); and Plowman et al.,Nature 366:473-75 (1993). Plowman et al. found that increased erbB4expression closely correlated with certain carcinomas of epithelialorigin, including breast adenocarcinomas. Diagnostic methods fordetection of human neoplastic conditions (especially breast cancers)that evaluate erbB4 expression are described in EP Patent ApplicationNo. 599,274.

The quest for the activator of the erbB2 oncogene has lead to thediscovery of a family of heregulin polypeptides. In humans, theheregulin polypeptides characterized thus far are derived from alternatesplicing of a single gene which was mapped to the short arm ofchromosome 8 by Lee and Wood, Genomics 16:790-91 (1993).

Holmes et al. isolated and cloned a family of polypeptide activators forthe ErbB-2 receptor which they called heregulin-α (HRG-α), heregulin-β1(HRG-β1), heregulin-β2 (HRG-β2), and heregulin-β3 (HRG-β3). See Holmeset al., Science 256:1205-10 (1992); WO 92/20798; and U.S. Pat. No.5,367,060. These researchers demonstrated the ability of the purifiedheregulin polypeptides to activate tyrosine phosphorylation of theErbB-2 receptor in MCF7 breast tumor cells. Furthermore, the mitogenicactivity of the heregulin polypeptides on SK-BR-3 cells (which expresshigh levels of the ErbB-2 receptor) was also demonstrated.

Heregulins are large multi-domain proteins that are typically expressedas "pro-heregulins." Pro-heregulins have been shown to undergoproteolytic processing to a mature soluble form (usually of about 44-45kDa). Processing has been shown to occur intracellularly or at the cellsurface. Domains in the soluble form include (in order from the N- tothe C-terminus) an immunoglobulin homology (Ig-like) domain, a spacerregion rich in glycosylation sites, and a domain similar to a domainfound in EGF that is sufficient for ErbB receptor binding andactivation.

See Barbacci, et al., J. Biol. Chem. 270:9585-89 (1995).

The heregulin EGF-like domains are characterized by substantialstructural similarities to (Jacobsen et al., Biochemistry 35:3402-17[1996]), and limited sequence homology with, EGF residues 1-48 (Holmes,et al., supra). Functional similarities between the heregulin EGF-likedomains and EGF have been established by data showing that blocks of EGFsequence substituted into heregulin-β1 do not impair binding to cellsco-expressing ErbB-3 and ErbB-2. Barbacci et al., supra.

While heregulins are substantially identical in the first 213 amino acidresidues, they are classified into two major types, α and β, based ontwo EGF-like domains that differ in their C-terminal portions. Forexample, the heregulin-α EGF-like domain differs from that of theβ1-isoform by nine substitutions near the C-terminus. The β-isoform hasbeen reported to bind ErbB receptors with approximately eight to 10-foldhigher affinity than the α-isoform. Wen et al., Mol. Cell. Biol.14:1909-19 (1994).

The solution structure of the heregulin-α EGF domain has recently beendetermined at high resolution by NMR. Jacobsen et al., supra; Nagata etal., EMBO J. 13, 3517-3523 (1994). The salient features of this domaininclude (1) an N-terminal subdomain containing a central three-strandedβ-sheet with an intermittent helix and (2) a smaller C-terminalsubdomain that contains a short stretch of β-sheet. The EGF domain isstabilized by three disulfide bonds, two in the N-terminal subdomain andone in the C-terminal subdomain. The pairing of the six correspondingcysteine residues is conserved in EGF-like domains from all heregulinsand from EGF.

The 44 kDa neu differentiation factor (NDF), which is the rat equivalentof human HRG, was first described by Peles et al., Cell, 69:205-16(1992), and Wen et al., Cell, 69:559-72 (1992). Like the human heregulinpolypeptides, NDF has an Ig-like domain followed by an EGF-like domainand lacks a N-terminal signal peptide. Subsequently, Wen et al. carriedout "exhaustive cloning" to extend the family of NDFs. Wen et al., Mol.Cell. Biol., 14:1909-19 (1994). This work revealed six distinctfibroblastic pro-NDFs. Adopting the nomenclature of Holmes et al., theNDFs were classified as either α or β polypeptides based on thesequences of the EGF-like domains. Isoforms 1 to 4 are characterized onthe basis of a variable region between the EGF-like domain andtransmembrane domain. Also, isoforms a, b and c are defined based onvariable-length cytoplasmic domains. These researchers conclude thatdifferent NDF isoforms are generated by alternative splicing and performdistinct tissue-specific functions. See also EP 505 148; WO 93/22424;and WO 94/28133 (discussing NDF).

Falls et al., Cell 72:801-815 (1993) describe another member of theheregulin family which they call "acetylcholine receptor inducingactivity (ARIA) polypeptide." The chicken-derived ARIA polypeptidestimulates synthesis of muscle acetylcholine receptors. See WO 94/08007.ARIA is a β-type heregulin and lacks the entire spacer region betweenthe Ig-like domain and EGF-like domain of HRG-α and HRGβ1-β3.

Marchionni et al., Nature 362:312-318 (1993) identified severalbovine-derived proteins that they call "glial growth factors (GGFs)."These GGFs share the Ig-like domain and EGF-like domain with the otherheregulin proteins described above, but also have an amino-terminalkringle domain. GGFs generally do not have the complete spacer regionbetween the Ig-like domain and EGF-like domain. Only one of the GGFs,GGFII, has an N-terminal signal peptide. See also WO 92/18627; WO94/00140; WO 94/04560; WO 94/26298; WO 95/32724 (describing GGFs anduses thereof).

Ho et al. describe another member of the heregulin family called"sensory and motor neuron-derived factor (SMDF)." Ho et al., J. Biol.Chem. 270:14523-32 (1995). This protein has an EGF-like domaincharacteristic of all other heregulin polypeptides but a distinctN-terminal domain. In addition, SMDF lacks both the Ig-like domain andthe spacer region found in other heregulin polypeptides. Another featureof SMDF is the presence of two stretches of hydrophobic amino acids nearthe N-terminus.

While the heregulin polypeptides were first identified based on theirability to activate the ErbB-2 receptor (see Holmes et al., supra), ithas been discovered that certain ovarian cells expressing neu (erbB2)and neu-transfected fibroblasts did not bind or crosslink to NDF, nordid they undergo tyrosine phosphorylation in response to NDF. Peles etal., EMBO J. 12:961-71 (1993). This finding indicated that anothercellular component was necessary for conferring full heregulinresponsiveness.

Carraway et al. subsequently demonstrated that ¹²⁵ I-rHRG-β1 177-244bound to NIH-3T3 fibroblasts stably transfected with bovine erbB3 butnot to non-transfected parental cells. These researchers also expressedbovine ErbB-3 receptor in insect cells and showed that HRG-β1 177-244bound to a preparation of ErbB-3 receptor solubilized from these cells.They concluded that ErbB-3 is a receptor for heregulin and mediatesphosphorylation of intrinsic tyrosine residues as well asphosphorylation of ErbB-2 receptor in cells that express both receptors.Carraway et al., J. Biol. Chem. 269:14303-06 (1994). Sliwkowski et al.found that cells transfected with erbB3 alone show low affinities forheregulin, whereas cells transfected with both erbB2 and erbB3 showhigher affinities. Sliwkowski et al., J. Biol. Chem. 269:14661-65(1994).

Plowman and his colleagues have similarly studied ErbB-4/ErbB-2 receptoractivation. They expressed the ErbB2 receptor alone, the ErbB4 receptoralone, or the two receptors together in human T lymphocytes anddemonstrated that heregulin is capable of stimulating tyrosinephosphorylation of ErbB-4, but could only stimulate ErbB-2phosphorylation in cells expressing both receptors. Plowman et al.,Nature 336:473-75 (1993).

These observations are consistent with the "receptor cross-talking"concept described previously by Kokai et al., Cell 58:287-92 (1989),Stern et al., EMBO J. 7:995-1001 (1988), and King et al., 4:13-18(1989). These researchers found that binding of EGF to the EGFR resultedin activation of the EGFR kinase domain and cross-phosphorylation of theErbB-2 receptor. This is believed to be a result of ligand-inducedreceptor heterodimerization and the concomitant cross-phosphorylation ofthe receptors within the heterodimer. Wada et al., Cell 61:1339-47(1990).

Thus, the ErbB receptors are believed to be activated by ligand-inducedreceptor dimerization. Specifically, heregulins can bind separately toErbB-3 and ErbB-4 receptors, but not to the ErbB-2 receptor. However,ErbB-2 is required for signalling, and heterodimers containing ErbB-2 incombination with ErbB-3 or ErbB-4 bind heregulins with higher affinitythan homodimers containing ErbB-3 or ErbB-4. Plowman et al., Nature366:473-75 (1993); Sliwkowski et al., J. Biol. Chem. 269:14661-65(1994).

The biological activities of heregulins have been investigated byseveral groups. For example, Holmes et al. (supra) found that heregulinexerts a mitogenic effect on mammary cell lines (such as SK-BR-3 andMCF-7). Lewis et al. reported that heregulin-β1 stimulated proliferationand enhanced colony formation in soft agar in a number of human breastand ovarian tumor cell lines. Lewis et al., Cancer Research 56:1457-65(1996). These researchers also showed that ErbB-2 is a critical mediatorof heregulin responsiveness.

Pinkas-Kramarski et al. found that NDF (rat heregulin) is expressed inneurons and glial cells in embryonic and adult rat brain and primarycultures of rat brain cells, and suggested that NDF may act as asurvival and maturation factor for astrocytes. Pinkas-Kramarski et al.,PNAS USA 91:9387-91 (1994). Danilenko et al. reported that theinteraction of NDF and the ErbB-2 receptor is important in directingepidermal migration and differentiation during wound repair. Danilenkoet al., Abstract 3101, FASEB 8(4-5):A535 (1994).

Meyer and Birchmeier analyzed expression of mouse heregulin duringembryogenesis and in the perinatal animal using in situ hybridizationand RNase protection experiments. Meyer and Birchmeier, PNAS USA91:1064-68 (1994). These authors conclude, based on expression of thismolecule, that heregulin plays a role in vivo as a mesenchymal andneuronal factor. Their findings also indicated that heregulin functionsin the development of epithelia.

Falls et al. (supra) found that chicken ARIA plays a role in myotubedifferentiation, namely affecting the synthesis and concentration ofneurotransmitter neurons. Corfas and Fischbach demonstrated that ARIAalso increases the number of sodium channels in chick muscle. Corfas andFischbach, J. Neuroscience 13:2118-25 (1993).

Bovine GGFs have been reported to be mitogenic for Schwann cells. See,e.g., Brockes et al., J. Biol. Chem. 255:8374-77 (1980); Lemke andBrockes, J. Neurosci. 4:75-83 (1984); Brockes et al., J. Neuroscience4:75-83 (1984); Brockes et al., Ann. Neurol. 20:317-22 (1986); Brockes,Methods in Enzym. 147:217-225 (1987); Marchionni et al., supra. Schwanncells provide myelin sheathing around the axons of myelinated neuronsand thus play an important role in the development, function andregeneration of peripheral nerves. The implications of this role from atherapeutic standpoint have been addressed by Levi et al., J.Neuroscience 14:1309-19 (1994). Levi et al. discussed the potential forconstruction of a cellular prosthesis including Schwann cells that couldbe transplanted into areas of damaged spinal cord. Methods for culturingSchwann cells ex vivo have been described. See WO 94/00140; Li et al.,J. Neuroscience 16:2012-19 (1996).

GGFII has been shown to be mitogenic for subconfluent quiescent humanmyoblasts, and differentiation of clonal human myoblasts in thecontinuous presence of GGFII results in greater numbers of myotubesafter six days of differentiation. Sklar et al., J. Cell Biochem., Abst.W462, 18D, 540 (1994); see also WO 94/26298.

The relationship between the structure and function of new proteins canbe investigated using any of a variety of available mutational analysistechniques. Examples of such techniques include alanine scanningmutagenesis and phagemid display. Alanine scanning can be used toidentify active residues (i.e., residues that have a significant effecton protein function) in a protein or protein domain. For example,Cunningham and Wells used alanine scanning to identify residues in humangrowth hormone that were important for binding its receptor. Cunninghamand Wells, Science 244:1081-85 (1989). In alanine scanning, a geneencoding the protein or domain to be scanned is inserted into anexpression vector, and mutagenesis is carried out to generate a seriesof vectors that encode proteins or domains in which sequential residuesare converted to alanine. The encoded proteins or domain are expressedfrom these vectors, and the activities of the alanine-substitutedvariants are then tested to identify those with altered activity. Analteration in activity indicates that the residue at thealanine-substituted position is an active residue.

Phagemid display was developed to allow the screening of a large numberof variant polypeptides for a particular binding activity. Smith andParmley demonstrated that foreign peptides can be "displayed"efficiently on the surface of filamentous phage by inserting short genefragments into gene III of the fd phage. Smith, Science 228:1315-17(1985); Parmley and Smith, Gene 73:305-18 (1985). The gene III coatprotein is present in about five copies at one end of the phageparticle. The modified phage were termed "fusion phage" because theydisplayed the foreign peptides fused to the gene III coat protein. Aseach fusion phage particle displayed approximately five copies of thefusion protein, this mode of phage display was termed "polyvalentdisplay."

Scott et al. and Cwirla et al. showed that fusion phage libraries couldbe screened by sequential affinity selections known as "panning." Scottet al., Science 249:386-90 (1990); Cwirla et al., PNAS USA 87:6378-82(1990). However, early efforts to select high affinity fusion phagefailed, presumably due to the polyvalence of the phage particles. Thisproblem was solved with the development of a "monovalent" phage displaysystem in which the fusion protein is expressed at a low level from aphagemid and a helper phage provides a large excess of wild-type coatprotein. Bass et al., Proteins 8:309-14 (1990); Lowman et al., Biochem.30:10832-38 (1991). Monovalent phage display can be used to generate andscreen a large number of variant polypeptides to isolate those that bindwith high affinity to a target of interest.

SUMMARY OF THE INVENTION

The present invention provides a heregulin variant having an amino acidsequence not found in nature and the ability to bind an ErbB receptor.In one embodiment, the variant has an amino acid substitution at aselected residue corresponding to a residue of 645-amino acid nativehuman heregulin-β1 selected from the group consisting of:

S177, H178, L179, V180, K181, E184, E186, K187, T188, V191, N192, G193,G194, E195, M198, V199, K200, D201, N204, P205, S206, R207, Y208, L209,K211, P213, N214, E215, T217, G218, D219, Q222, N223, Y224, S228, andF229.

In a variation of this embodiment, the amino acid substitution is not areplacement of the selected residue with an epidermal growth factor(EGF) residue corresponding to the selected residue.

The heregulin variant can be a variant of any member of the heregulinfamily from any species. In one embodiment, the heregulin variant is avariant of a human heregulin, such as, for example, human heregulin-β1.The invention provides a human heregulin-β1 variant including an aminoacid substitution selected from the group consisting of:

S177W; H178S, E, R, or A; V180Q, I, or E; K181P or A; A183G; E184V, W,K, R, G, or N; K185E, S, Q, or G; E186R; K187E or A; T188Q; E195Q;F197Y; M198R or K; K200R; D201T or I; P205T or Y; S206K, H, G, P, or R;R207Y; Y208R or L; L209M or G; K211R; P213S, T, N, or K; N214L, K, S, orE; F216M; N223H or W; and M226I.

In a variation of this embodiment, the heregulin variant includes setsof amino acid substitutions selected from this group. Some heregulinvariants of the invention having sets of amino acid substitutionsexhibit at least a 50-fold increase in ErbB-3 receptor affinity, whichis also accompanied by an increase in ErbB-4 receptor affinity.

The invention also includes a heregulin variant that has a greaterspecificity for the ErbB-4 receptor, relative to the ErbB-3 receptor,than the heregulin from which the heregulin variant is derived. In oneembodiment, this heregulin variant has an amino acid substitution at aselected residue corresponding to a residue of 645-amino acid nativehuman heregulin-β1 selected from the group consisting of H178, L179, andR207.

In another embodiment, a heregulin variant having a greater specificityfor the ErbB-4 receptor, relative to the ErbB-3 receptor, has a deletionof amino acid residues corresponding to residues S228 to K231 of645-amino acid native human heregulin-β1 and a substitution of a singlemethionine for the deleted residues.

In addition to including one or more of the mutations disclosed herein,the heregulin variant can have one or more other modifications, such asan amino acid substitution, an insertion of at least one amino acid, adeletion of at least one amino acid, or a chemical modification. Forexample, the invention provides a heregulin variant that is a fragment.In a variation of this embodiment, the fragment includes residuescorresponding to a portion of human heregulin-β1 extending from aboutresidue 175 to about residue 230 (i.e., the EGF-like domain). In afurther variation of this embodiment, the fragment includes residuescorresponding to a portion of human heregulin-β1 extending from aboutresidue 175 to about residue 245.

One aspect of the invention is a method for producing such a modifiedheregulin variant. The modification(s) is selected so that the modifiedheregulin variant retains the ability to an ErbB receptor.

In addition to a heregulin variant, the invention provides a relatednucleic acid molecule, vector, and host cell. The invention alsoprovides a method of producing a heregulin variant in which a host cellcontaining an expression vector capable of expressing the heregulinvariant is cultured under conditions that allow expression of theheregulin variant, and the heregulin variant then recovered from theculture.

Other aspects of the invention relate to various uses of a heregulinvariant. For example, the invention provides a method for activating anErbB receptor in which the heregulin variant is contacted with a cellthat expresses an ErbB receptor. The heregulin variant can be contactedwith cells in culture, for example, to promote ex vivo survival,proliferation, or differentiation of cells, such as glial, Schwann, ormuscle cells.

Alternatively, the heregulin variant can be combined with apharmaceutically acceptable carrier and used to treat one of a widerange of cancers as well as diseases and disorders affecting the nervoussystem, musculature, and epithelia. Thus, the present invention providesa pharmaceutical composition and a treatment method.

The invention also includes a method of determining whether a samplecontains an ErbB receptor that binds a heregulin. In particular, aheregulin variant is contacted with a sample, and specific bindingbetween the heregulin variant and a component of the sample isdetermined as an indication of the presence and/or amount of ErbBreceptor(s) present in the sample.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an alignment between the amino acid sequences in theEGF-like domains of a number of heregulin polypeptides and the aminoacid sequence of the human heregulin-β1 EGF-like domain ("betal"). Thealigned sequences are as follows: human heregulin-α (alpha), humanheregulin-β2 and -β3 (beta2 and beta3), neu differentiation factors a2and b1 to b4 (ndfa2 and ndfb1-4), glial growth factor II (ggf), sensoryand motor neuron-derived factor (smdf), human heregulin-γ, andacetylcholine receptor inducing activity polypeptide (aria). The numbershown for each sequence is the residue number of the first amino acidshown, as numbered from the N-terminus of the native polypeptide whosesequence is shown. "#" indicates the differences between the α- andβ-type EGF-like domains.

FIG. 2 shows the results of an alanine scan of the heregulin-β1 EGF-likedomain (heregulin-β1 residues 177-228). Individual amino acids in thisdomain were mutated to alanine and displayed monovalently on phage asgIII fusion proteins, as described in Example 2. The histogram shows thechange in binding affinity of each alanine variant for ErbB-3 and ErbB-4receptor-Ig fusions (ErbB-3-Ig and ErbB-4-Ig), as measured by phageELISA. The X axis lists each amino acid that was changed to alanine andits position. The Y axis is the ratio of the EC₅₀ for each variant tothe EC₅₀ for the wild-type heregulin-β1 EGF-like domain, also displayedon phage. The EC₅₀ was calculated as the concentration of solublereceptor fusion required to displace 50% of the total amount of phagebound to immobilized receptor fusion. ErbB-3 binding results are shownwith black bars, and ErbB-4 binding results are shown with white bars.

FIG. 3 shows the amino acids selected for binding to ErbB-3-Ig at eachposition in the heregulin-β1 EGF-like domain (heregulin-β1 residues177-228) randomized in the phage display studies described in Example 3.The length of the bars indicates the frequency of occurrence of aparticular amino acid at each position in the variants from phagedisplay libraries A-E and G-I for which sequences were determined (i.e.,a longer bar indicates a higher frequency). Twelve clones were sequencedfrom each library, although in library H, only one clone of the twelverepresented a variant having mutations in the desired randomizationwindow (see Example 3). "WT" indicates the wild-type amino acid sequenceof the heregulin-β1 EGF-like domain.

FIG. 4 shows the amino acid substitutions in the EGF-like domains ofcombination variants described in Example 3. The amino acid sequence ofthe wild-type heregulin-β1 EGF-like domain (HRG8), a variant of thisdomain containing a deletion of heregulin-β1 residues 202-204 (HRG63),and the analogous domain in EGF are shown on top. The residue numberingfor the portion of the heregulin-β1 amino acid sequence shown isindicated above this sequence (numbered from the N-terminus of nativehuman heregulin-β1). The residue numbering for the portion of the EGFamino acid sequence shown is indicated below this sequence (numberedfrom the N-terminus of native human EGF). A "." indicates a residue thatis identical to the wild-type residue at the particular position. A "-"indicates the absence of a residue.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides heregulin variants having one or moreamino acid substitutions at selected residues. Included within the scopeof the invention are variants of human heregulins, and, in particular,variants of human heregulin-β1. A heregulin variant according to theinvention can have a single amino acid substitution at a selectedresidue or combinations of such substitutions.

In addition to the amino acid substitutions specified herein, heregulinvariants according to the invention can have further modifications,including, for example, deletions of amino acids. In one embodiment, aheregulin variant has N- and C-terminal deletions, leaving only aminoacids corresponding to "the minimal EGF-like domain," which issufficient for binding and activation of an ErbB receptor.

A heregulin variant of the invention is capable of binding an ErbBreceptor, such as ErbB-3 or ErbB-4. In one embodiment, the variant hasan enhanced affinity for an ErbB receptor compared to the affinity ofthe most homologous native heregulin. In addition to ErbB receptorbinding, the heregulin variant can possess one or more other biologicalactivities of a native heregulin.

The invention also provides nucleic acid molecules, vectors, and hostcells related to the heregulin variants. A nucleic acid molecule of theinvention encodes, or is complementary to a nucleic acid moleculeencoding, a heregulin variant of the invention or a fragment thereof.The nucleic acid molecule can be double- or single-stranded DNA or RNA.A nucleic acid molecule of the invention can be inserted into anappropriate vector for propagation and/or expression of an encodedheregulin variant. Such vectors are introduced into suitable hosts, forexample, to allow recombinant production of a heregulin variant.

The heregulin variants of the invention are useful in a variety oftherapeutic and non-therapeutic applications. In particular, heregulinvariants can be used in treating cancer and various diseases anddisorders of the nervous system, musculature, and epithelia.Accordingly, the invention encompasses a pharmaceutical compositionincluding a heregulin variant and related treatment methods.

Heregulin variants can also be employed in a variety of non-therapeuticapplications, such as cell culture methods and diagnostic methods. Forexample, heregulin variants can be used to promote the ex vivo survival,proliferation, or differentiation of cells, including glial and musclecells. In an exemplary diagnostic application, heregulin variants areemployed in the diagnosis of a cancer characterized by erbB (e.g.,erbB2) overexpression. Accordingly, the invention also includes kitsuseful in practicing the above-described methods.

DEFINITIONS

As used herein, the following words or phrases have the definitionsindicated below, unless otherwise indicated.

The terms "amino acid" and "residue" are used interchangeably herein.

The term "wild-type amino acid" or "wild-type residue" means the aminoacid present at a given position(s) in a native polypeptide.

Amino acids are denoted herein by the standard three-letter orone-letter code.

Residues in two or more polypeptides are said to "correspond" if theresidues occupy an analogous position in the polypeptide structures. Asis well known in the art, analogous positions in two or morepolypeptides can be determined by aligning the polypeptide sequencesbased on amino acid sequence or structural similarities. Those skilledin the art understand that it may be necessary to introduce gaps ineither sequence to produce a satisfactory alignment. For example,residues in human EGF that correspond to residues in human heregulin-β1are shown in an alignment between the amino acid sequence of theheregulin-β1 EGF-like domain (heregulin-β1 residues 177-228) and theanalogous EGF domain (EGF residues 1-48) in FIG. 4.

Residues in two or more heregulins are said to "correspond" if theresidues are aligned in the best sequence alignment. The "best sequencealignment" between two polypeptides is defined as the alignment thatproduces the largest number of aligned identical residues. The bestsequence alignment for a number of heregulin polypeptides is shown inFIG. 1.

Residue positions in heregulin-β1 are designated herein by thethree-letter or one-letter code for the amino acid, followed by theposition number, as numbered from the N-terminus of native humanpro-heregulin-β1 (which is 645 amino acids in length). E.g., the serineat position 177 of heregulin-β1 is denoted "Ser177" or "S177."

Hereinafter, unless otherwise indicated, residue positions in aheregulin, heregulin variant, or related protein, such as EGF, arespecified herein with reference to the amino acid numbering of nativehuman heregulin-β1. For example, a heregulin-β1 variant can have aN-terminal deletion of residues 1-176. The first amino acid in thisvariant is identified herein as "the residue corresponding to Ser177 of645-amino acid human heregulin-β1" because the first residue of theheregulin variant and Ser177 of heregulin-β1 are aligned in the bestalignment between the two polypeptides.

Example 3 discloses heregulin-β1 variants containing residuescorresponding to residues 177 to 228 of heregulin-β1, which is termed"the minimal EGF-like domain." For these variants, residue numbers alsoare expressed, in parentheses, in terms of the position of the residuein the minimal EGF-like domain (hereinafter "heregulin-β1 EGF" or"HRG-β1 EGF"), i.e., residues 1-52. Residue positions numbered accordingto native human heregulin-β1 can be converted to residue positions inthe minimal EGF-like domain by subtracting 176 from the former positionnumber. E.g., for heregulin-β1 Ser177, subtracting 176 from 177 gives 1,and thus heregulin-β1 EGF Ser1 identifies the same position asheregulin-β1 Ser177. The same numbering system is used in Example 4.

Amino acid substitutions are indicated by listing the residue positionfollowed by the code for the amino acid substituted into the heregulinpolypeptide. Thus, a substitution of alanine at Ser177 of heregulin-β1is expressed as "heregulin-β1 Ser177Ala," "Ser177Ala", or "S177A." Inthis example, serine is the "replaced amino acid," and alanine is the"replacement amino acid."

As used to describe two amino acid sequences, the term "homologous"indicates that the amino acid sequences have some degree of amino acidsequence identity.

HEREGULIN VARIANTS

The present invention includes a heregulin variant. The term "heregulinvariant" means a polypeptide variant of a native heregulin. A nativeheregulin is defined as a polypeptide having the full-length amino acidsequence of any of the family of naturally occurring heregulinpolypeptides. This family encompasses pro-heregulins as well as thesoluble forms of these proteins. The invention is exemplified withvariants of human heregulin-β1. See Examples 1-3. However, the heregulinfamily encompasses any naturally occurring polypeptide having anEGF-like domain that has at least 70 percent sequence identity with theEGF-like domain of heregulin-β1 when these domains are aligned in thebest alignment. Thus, a native heregulin can be from any species and oneof a number of naturally occurring isoforms or allelic forms. Exemplaryheregulin polypeptides include neu differentiation factors, glial growthfactors, sensory and motor neuron-derived factor, and acetylcholinereceptor inducing activity polypeptide.

In one embodiment, the heregulin variant is a variant of a mammalianheregulin. In a variation of this embodiment, the heregulin variant is avariant of a human heregulin. Examples of human heregulins includeheregulin-α (HRG-α), heregulin-β1 (HRG-β1), heregulin-β2 (HRG-β2),heregulin-β3 (HRG-β3), and heregulin-γ (HRG-γ).

A heregulin variant according to the invention has an amino acidsequence not found in nature in which a wild-type residue in a nativeheregulin is replaced with a different residue. This amino acidsubstitution is at one or more selected residues corresponding to aresidue of native human heregulin-β1. The selected residue(s) is chosenfrom the following group:

S177, H178, L179, V180, K181, E184, E186, K187, T188, V191, N192, G193,G194, E195, M198, V199, K200, D201, N204, P205, S206, R207, Y208, L209,K211, P213, N214, E215, T217, G218, D219, Q222, N223, Y224, S228, andF229.

In one embodiment, however, the amino acid substitution is not areplacement of the selected residue with an EGF residue corresponding tothe selected residue. Substitution of any of the above residues inheregulin-β1 with alanine has at least a two-fold effect on affinity forthe ErbB-3 or ErbB-4 receptors, as determined by phage ELISA. SeeExample 2.

In one embodiment, the selected residue is chosen from the followinggroup:

S177, H178, L179, E186, K187, T188, V191, N192, G193, G194, E195, R207,L209, K211, P213, N214, T217, G218, Q222, Y224, and F229.

Substitution of the any of these residues in heregulin-β1 with alaninehas at least a five-fold effect on affinity for the ErbB-3 or ErbB-4receptors, as determined by phage ELISA. See Example 2.

In a variation of this embodiment, the selected residue is chosen fromthe following group:

H178, L179, K187, N192, G193, G194, E195,

R207, K211, T217, G218, Q222, and Y224.

Substitution of the any of these residues in heregulin-β1 with alaninehas at least a 10-fold effect on affinity for the ErbB-3 or ErbB-4receptors, as determined by phage ELISA. See Example 2.

Generally, if function is to be preserved at a position selected forsubstitution, the residue used to replace the selected residue is notsubstantially different in character from the wild-type residue, i.e.,the amino acid substitution is a conservative substitution. Amino acidscan be grouped according to character as shown in Table 1.

                  TABLE 1                                                         ______________________________________                                        Groups of Amino Acids                                                          Having Similar Character                                                       Group         Character      Amino Acids                                    ______________________________________                                        a           positively charged                                                                           Lys, Arg, His                                        b negatively charged Asp, Glu                                                 c amide Asn, Gln                                                              d aromatic Phe, Tyr, Trp                                                      e hydrophobic Pro, Gly,                                                         Ala, Val,                                                                     Leu, Ile, Met                                                               f uncharged Ser, Thr                                                           hydrophilic                                                                ______________________________________                                    

To preserve function, therefore, the residue used to replace thewild-type residue is usually selected from the same group or a relatedgroup. In addition, serine or alanine can be used to replace most otherresidues. Table 2 shows conservative substitutions for each amino acid,identifying related groups for each and indicating which amino acids canbe replaced with serine or alanine. Table 2 also shows preferred aminoacid substitutions.

                  TABLE 2                                                         ______________________________________                                        Conservative Amino Acid Substitutions                                                     Replacement Amino   Preferred                                       AA* Acid Selected From Substitutions                                        ______________________________________                                        Ala     e.sup.# : Pro, Gly, Ala, Val, Leu, Ile, Met                                                       Ser                                                  f: Ser, Thr                                                                  Arg a: Lys, Arg, His Lys                                                       Ser, Ala Ser, Ala                                                            Asn a: Lys, Arg, His                                                           c: Asn, Gln Gln                                                               Ser, Ala Ser, Ala                                                            Asp b: Asp, Glu Glu                                                            c: Asn, Gln                                                                   Ser, Ala Ser, Ala                                                            Cys e: Pro, Gly, Ala, Val, Leu, Ile, Met Ala                                   f: Ser, Thr Ser                                                              Gln a: Lys, Arg, His                                                           c: Asn, Gln Asn                                                               Ser, Ala Ser, Ala                                                            Glu b: Asp, Glu Asp                                                            c: Asn, Gln                                                                   Ser, Ala Ser, Ala                                                            Gly e: Pro, Gly, Ala, Val, Leu, Ile, Met Pro, Ala                              f: Ser, Thr                                                                  His a: Lys, Arg, His Arg                                                       Ser, Ala Ser, Ala                                                            Ile e: Pro, Gly, Ala, Val, Leu, Met Ala, Val, Leu                             Leu e: Pro, Gly, Ala, Val, Ile, Met Ala, Val, Ile                             Lys a: Lys, Arg, His Arg                                                       Ser, Ala Ser, Ala                                                            Met e: Pro, Gly, Ala, Val, Leu, Ile, Met Ala, Val, Leu, Ile                   Phe a: Lys, Arg, His                                                           d: Phe, Tyr, Trp Tyr                                                          Ala, Val, Leu, Ile Ala, Val, Leu, Ile                                        Pro a: Lys, Arg, His                                                           d: Phe, Tyr, Trp Phe                                                          Gly, Ala Gly, Ala                                                            Ser a: Lys, Arg, His Thr                                                       f: Ser, Thr                                                                   Ala Ala                                                                      Thr a: Lys, Arg, His                                                           f: Ser, Thr Ser                                                               Ala Ala                                                                      Trp d: Phe, Tyr, Trp Phe                                                       Ala Ala                                                                      Tyr d: Phe, Tyr, Trp Phe,                                                      Ala, Val, Leu, Ile Ala, Val, Leu, Ile                                        Val e: Pro, Gly, Ala, Val, Leu, Ile, Met Leu, Ile,                             Ser, Ala Ser, Ala                                                          ______________________________________                                         *Wild-type amino acid.                                                        .sup.# Lowercase letters refer to the groups of amino acids in Table 1.  

In one embodiment, the heregulin variant is a variant of humanheregulin-β1 and includes an amino acid substitution(s) chosen from thefollowing group:

S177W; H178S, E, R, or A; V180Q, I, or E; K181P or A; A183G, T, or D;E184V, W, K, R, G, or N; K185E, S, Q, or G; E186R; K187E or A; T188Q;E195Q; F197Y; M198R or K; K200R; D201T or I; P205T or Y; S206K, H, G, P,or R; R207Y; Y208R or L; L209M or G; K211R; P213S, T, N, or K; N214L, K,S, or E; F216M; N223H or W; and M226I.

Another aspect of the invention is a variant of human heregulin-β1including a set of amino acid substitutions, such as any of theheregulin variants described in Example 3. The set of amino acidsubstitutions is chosen from the group indicated below (the variantnumber from Example 3 is shown in the left-hand column, followed by theset of amino acid substitutions for that variant):

B5: A183G, E184W, K185D, E186R, K187E, T188G, M226I;

B10: A183D, E184K, K185S, E186R, K187E, T188G, M226I;

D1: F197Y, M198K, K200R, D201I, M226I;

E2: P205Y, S206G, R207Y, Y208L, L209M;

E3: P205Y, S206R, R207Y, Y208R, L209M, M226I;

E6: P205T, S206H, R207Y, Y208R, L209M;

E8: P205T, S206K, R207Y, Y208R, L209G;

I1: N223W, M226I;

I2: N223H, M226I;

HRG37: S177W, H178E, K181P, A183G, E184W, K185D, E186R, K187E, T188G,M226I;

HRG48: P205Y, S206G, R207Y, Y208L, L209M, M226I;

HRG53: A183G, K185E, E186R, K187E, T188G, F197Y, M198R, D201T;

HRG54: A183G, K185E, E186R, K187E, T188G, P205Y, S206G, R207Y, Y208L,L209M;

HRG55: A183G, K185E, E186R, K187E, T188G, F197Y, M198R, D201T, P205Y,S206G, R207Y, Y208L, L209M;

HRG56: A183G, K185E, E186R, K187E, T188G, M226I;

HRG57: F197Y, M198R, D201T, P205Y, S206G, R207Y, Y208L, L209M;

HRG58: F197Y, M198R, D201T, P205Y, S206G, R207Y, Y208L, L209M, M226I;

HRG59: F197Y, M198R, D201T, M226I;

HRG60: A183G, K185E, E186R, K187E, T188G, F197Y, M198R, D201T, M226I;

HRG61: A183G, K185E, E186R, K187E, T188G, P205Y, S206G, R207Y, Y208L,L209M, M226I;

HRG62: A183G, K185E, E186R, K187E, T188G, F197Y, M198R, D201T, P205Y,S206G, R207Y, Y208L, L209M, M226I;

HRG71: F197Y, M198R, D201T, P205Y, S206G, R207Y, Y208L, L209M, N223H,M226I; and

HRG73: A183G, K185E, E186R, K187E, T188G, F197Y, M198R, D201T, P205Y,S206G, R207Y, Y208L, L209M, N223H, M226I.

Each of these sets of amino acid substitutions produces at least afive-fold increase in ErbB-3 receptor affinity, as determined by phageELISA. See Example 3.

In a variation of this embodiment, the set of amino acid substitutionsis chosen from the following group:

B5: A183G, E184W, K185D, E186R, K187E, T188G, M226I;

E2: P205Y, S206G, R207Y, Y208L, L209M;

I2: N223H, M226I;

HRG48: P205Y, S206G, R207Y, Y208L, L209M, M226I;

HRG53: A183G, K185E, E186R, K187E, T188G, F197Y, M198R, D201T;

HRG56: A183G, K185E, E186R, K187E, T188G, M226I;

HRG57: F197Y, M198R, D201T, P205Y, S206G, R207Y, Y208L, L209M;

HRG58: F197Y, M198R, D201T, P205Y, S206G, R207Y, Y208L, L209M, M226I;

HRG59: F197Y, M198R, D201T, M226I;

HRG60: A183G, K185E, E186R, K187E, T188G, F197Y, M198R, D201T, M226I;

HRG61: A183G, K185E, E186R, K187E, T188G, P205Y, S206G, R207Y, Y208L,L209M, M226I;

HRG62: A183G, K185E, E186R, K187E, T188G, F197Y, M198R, D201T, P205Y,S206G, R207Y, Y208L, L209M, M226I;

HRG71: F197Y, M198R, D201T, P205Y, S206G, R207Y, Y208L, L209M, N223H,M226I; and

HRG73: A183G, K185E, E186R, K187E, T188G, F197Y, M198R, D201T, P205Y,S206G, R207Y, Y208L, L209M, N223H, M226I.

Each of these sets of amino acid substitutions produces at least a20-fold increase in ErbB-3 receptor affinity, as determined by phageELISA. See Example 3.

In another variation of this embodiment, the set of amino acidsubstitutions is chosen from the following group:

HRG58: F197Y, M198R, D201T, P205Y, S206G, R207Y, Y208L, L209M, M226I;

HRG60: A183G, K185E, E186R, K187E, T188G, F197Y, M198R, D201T, M226I;

HRG71: F197Y, M198R, D201T, P205Y, S206G, R207Y, Y208L, L209M, N223H,M226I; and

HRG73: A183G, K185E, E186R, K187E, T188G, F197Y, M198R, D201T, P205Y,S206G, R207Y, Y208L, L209M, N223H, M226I.

Each of these sets of amino acid substitutions produces at least a50-fold increase in ErbB-3 receptor affinity, as determined by phageELISA. See Example 3.

The invention also includes a heregulin variant that has a greaterspecificity for the ErbB-4 receptor, relative to the ErbB-3 receptor,than the heregulin from which the heregulin variant is derived. Suchenhanced ErbB-4 receptor specificity can be measured, for example, as alower variant:wild-type EC₅₀ ratio for binding to the ErbB-4 receptorthan for binding to ErbB-3 receptor. Generally, a heregulin varianthaving enhanced ErbB-4 receptor specificity has approximately wild-typeaffinity for the ErbB-4 receptor or better together with a significantlyreduced (i.e., at least about 5-10-fold) affinity for the ErbB-3receptor.

In one embodiment, a heregulin variant having enhanced ErbB-4 receptorspecificity has an amino acid substitution at a selected residuecorresponding to a residue of 645-amino acid native human heregulin-β1selected from the group consisting of H178, L179, and R207. For example,the invention provides a human heregulin-β1 variant including the aminoacid substitution H178E or R207P.

In another embodiment, a heregulin variant having enhanced ErbB-4receptor specificity has a deletion of amino acid residues correspondingto residues S228 to K231 of 645-amino acid native human heregulin-β1 anda substitution of a single methionine for the deleted residues. In avariation of this embodiment, the heregulin variant also includes theamino acid substitution H178L.

In addition to the above amino acid substitutions, the heregulin variantcan optionally have any of the following modifications, singly or incombination: (1) one or more additional amino acid substitutions; (2)one or more amino acids added to the N- or C-terminus of, or insertedwithin, the amino acid sequence of the heregulin variant; (3) one ormore amino acids deleted from the heregulin variant; and (4) one or morecovalent modifications of an amino acid in the heregulin variant.

Thus, the heregulin variant can be "substantially full-length," which,as used herein, means that the heregulin variant is at least 90% as longas the native heregulin to which the variant is most homologous.Alternatively, the heregulin variant can be a "fragment" that is lessthan 90% as long as the most homologous native heregulin. Heregulinvariants that are fragments are usually about 30 to about 100 aminoacids, more usually about 40 to about 60 amino acids, even more usuallyabout 45 to about 65 amino acids, and most usually about 50 amino acidsin length.

For example, the heregulin variant can include amino acids correspondingto "the minimal EGF-like domain." The minimal EGF-like domain is aportion of a native heregulin that is sufficient for binding andactivation of an ErbB receptor. In general, the minimal EGF-like domainis less than about 70 amino acids and usually less than about 60 aminoacids in length. As used herein with reference to human heregulin-β1,the minimal EGF-like domain extends from residues 177-228. Unlessotherwise indicated, "HRG-β1 EGF" refers to the minimal EGF-like domain.

Examples of suitable covalent modifications of a heregulin variantaccording to the invention include, but are not limited to, conjugationwith a detectable label, "pegylation," and conjugation with a cytotoxicagent. A heregulin variant can be conjugated to any of a wide variety ofavailable labels to produce a conjugate useful for detecting thepresence of ErbB receptors in a sample. Suitable labels include aradioisotope, a fluorescent label, and an enzyme label. Exemplaryradioisotope labels are ³⁵ S, ¹⁴ C, ¹²⁵ I, ³ H, and ¹³¹ I. Heregulinvariants can be conjugated to radioisotopes as described generally inCurrent Protocols in Immunology Vols. 1 & 2 (Coligen et al. ed., WileyPublishers).

Fluorescent labels suitable for conjugation to a heregulin variantinclude a rare earth chelate (a europium chelate), fluorescein,rhodamine, dansyl, Lissamine, phycoerythrin, and Texas Red, andderivatives thereof. Conjugates can be prepared as described, forexample, in Current Protocols in Immunology supra.

Various enzyme-substrate systems are available, and U.S. Pat. No.4,275,149 provides a review of some of these. In general, enzymes usefulin such systems catalyze a readily detectable chemical alteration of asubstrate. For example, the enzyme can catalyze a color change, whichcan be measured spectrophoto-metrically, or a change in fluorescence orchemiluminescence, which can be detected using a fluorometer orchemiluminometer, respectively. Exemplary enzyme labels include aluciferase, malate dehydrogenase, urease, a peroxidase, alkalinephosphatase, β-galactosidase, glucoamylase, lysozyme, a saccharideoxidase, a heterocyclic oxidase, lactoperoxidase, microperoxidase, andthe like. Heregulin variants can be conjugated to enzyme labels asdescribed generally in O'Sullivan et al., Methods in Enzym. 73:47-166(1981), and in Current Protocols in Immunology (supra). Suitablesubstrates for use with a given enzyme label are well known to thoseskilled in the art.

Another exemplary modification of a heregulin variant of the inventionis pegylation, which refers to the conjugation of one or morepolyethylene glycol (PEG) groups to the ε-amino group(s) of apolypeptide. Pegylation may be desired when the heregulin variant isintended for pharmaceutical use, as pegylation can increase in vivohalf-life and/or reduce immunogenicity and potential toxicity oftherapeutic proteins. See, e.g., Abuchowski et al., J. Biol. Chem.252:3582-86 (1977).

Conjugation of a heregulin variant with a cytotoxic agent produces atargeted cytotoxic agent that specifically binds cells expressingappropriate ErbE receptors on their surface. The term "cytotoxic agent"refers to a substance that inhibits or prevents the function of cellsand/or causes destruction of cells. The term includes, for example, aradioactive isotope (e.g., I, Y, Pr) and a chemotherapeutic agent.

A "chemotherapeutic agent" is defined herein as any chemical compounduseful in the treatment of cancer. The term "cancer" refers to thephysiological condition in mammals that is characterized by unregulatedcell growth. Examples of cancer include but are not limited to,carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More particularexamples of such cancers include squamous cell cancer, small-cell lungcancer, non-small cell lung cancer, gastric cancer, pancreatic cancer,glial cell tumors such as glioblastoma and neurofibromatosis, cervicalcancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breastcancer, colon cancer, colorectal cancer, endometrial carcinoma, salivarygland carcinoma, kidney cancer, renal cancer, prostate cancer, vulvalcancer, thyroid cancer, hepatic carcinoma and various types of head andneck cancer. Examples of chemotherapeutic agents include Adriamycin,Doxorubicin, 5-Fluorouracil (5-FU), Cytosine arabinoside (Ara-C),Cyclophosphamide, Thiotepa, Busulfan, Cytoxin, Taxol, Methotrexate,Cisplatin, Melphalan, Vinblastine, Bleomycin, Etoposide, Ifosfamide,Mitomycin C, Mitoxantrone, Vincristine, VP-16, Vinorelbine, Carboplatin,Teniposide, Daunomycin, Carminomycin, Aminopterin, Dactinomycin, aMitomycin, Nicotinamide, an Esperamicin, Melphalan and any relatednitrogen mustard, and an endocrine therapeutic (such asdiethylstilbestrol [DES], Tamoxifen, a leutinizing hormone releasinghormone-antagonizing drug, a progestin, an anti-progestin, etc.).

In addition to conjugation to a chemical compound, any of theabove-described heregulin variants can be modified by fusion to aheterologous polypeptide to produce a "chimeric heregulin variant."(Chimeric heregulin variants are also referred to herein as "fusionproteins.) Typically, the heterologous polypeptide is fused at the N- orC-terminus of the heregulin variant to preserve the biological activity(described further below) of the heregulin variant. However, theheterologous polypeptide can also be introduced into regions of theheregulin variant that are not critical for biological activity.Generally, chimeric heregulin variants are produced by recombinanttechniques. Examples of chimeric heregulin variants include a heregulinvariant fused to a "signal sequence," a "purification handle" and animmunoglobulin sequence.

A "signal sequence" is an amino acid sequence that directs the secretionof a polypeptide fused thereto from a cell expressing the chimericprotein. Thus, fusion of a heregulin variant to a signal sequencefacilitates recombinant production of the heregulin variant because thechimeric heregulin variant is secreted into the host cell culturemedium, from which the chimeric heregulin variant can be recovered withrelative ease.

A suitable signal sequence can be obtained from any protein that has asignal sequence and is typically (but not always) fused to theN-terminus of the heregulin variant. DNA encoding prokaryotic signalsequences can be obtained, for example, from lamB or ompF, MalE, PhoA,and other genes. A convenient prokaryotic signal sequence for practicingthe invention is the E. coli heat-stable enterotoxin II (STII) signalsequence.

A "purification handle" is a portion of a polypeptide that binds anotherpolypeptide, termed a "binding partner." The fusion of a purificationhandle to a heregulin variant confers on the variant the ability to bindthe binding partner, which facilitates purification of the resultantchimeric heregulin variant. Generally, the purification handle isselected so that the binding partner does not substantially cross-reactwith other components present in the mixture from which the chimericheregulin variant is to be purified. As used herein, the term "does notsubstantially cross-react" means that the affinity of the bindingpartner for the purification handle is at least about 20-fold, usuallyat least about 100-fold, more usually at least about 1000-fold, anyaffinity for any other components present in the mixture.

In one embodiment, the purification handle is an epitope recognized byan antibody, and the chimeric heregulin variant is therefore termed an"epitope-tagged heregulin variant." Suitable epitopes generally have atleast five amino acids, usually between about 10 and about 50 aminoacids, and more usually between about 10 and about 30 amino acids.

A chimeric molecule that includes a heregulin variant fused to animmunoglobulin sequence is termed "a heregulin variant immunoadhesin."In one embodiment, the immunoglobulin sequence is an immunoglobulinconstant domain. The immunoglobulin sequence in a heregulin variantimmunoadhesin can be obtained from IgG₁, IgG₂, IgG₃ or IgG₄ subtypes,IgA, IgE, IgD, or IgM. In one embodiment, the immunoglobulin sequence isobtained from IgG₁ or IgG₃.

Other examples of chimeric heregulin variants include heregulin variantsfused to thioredoxin, a "salvage receptor binding epitope," or acytotoxic polypeptide. Fusion of a heregulin variant with thioredoxinenhances expression and provides a purification handle that facilitatespurification using phenylarsine oxide, which can be covalently bound toa solid support, such as agarose. (Agarose functionalized withphenylarsine is available commercially as Thibond™ Resin from InvitrogenCorp., San Diego, Calif.) Exemplary thioredoxin-variant fusion proteinsare described in Example 2.

The term "salvage receptor binding epitope" refers to an epitope of theFc region of an IgG molecule (e.g., IgG₁, IgG₂, IgG₃, or IgG₄) thatincreases the in vivo serum half-life of the IgG. Salvage receptorbinding epitopes suitable for fusion to a heregulin variant accordingthe invention include any of the known salvage receptor bindingepitopes.

The term "cytotoxic polypeptide" refers to a polypeptide that inhibits acellular function or kills cells. Cytotoxic polypeptides suitable forfusion to a heregulin variant include an enzymatically active toxin ofbacterial, fungal, plant, or animal origin and fragments thereof and anoncogene product/tyrosine kinase inhibitor, such as a peptide thatinhibits binding of a tyrosine kinase to a SH2-containing substrateprotein (see WO 94/07913, for example).

In one embodiment, a chimeric heregulin variant includes a heregulinvariant fused to an enzyme that converts a "prodrug" to an active drug.Typically, the "prodrug" is a precursor or derivative form of acytotoxic drug that is less cytotoxic than the drug itself and iscapable of being enzymatically activated or converted to the cytotoxicdrug. The prodrugs of this invention include, but are not limited to, aphosphate-containing prodrug, a thiophosphate-containing prodrug, asulfate-containing prodrug, a peptide-containing prodrug, a D-aminoacid-modified prodrug, a glycosylated prodrug, a β-lactam-containingprodrug, a phenoxyacetamide-containing prodrug, aphenylacetamide-containing prodrug, 5-fluorocytosine, a 5-fluorouridineprodrug, and derivatives thereof. Examples of cytotoxic drugs that canbe derivatized to produce a prodrug for use in this invention include,but are not limited to, those chemotherapeutic agents described above.

A heregulin variant according to the invention is capable of binding anErbB receptor. The term "ErbB receptor" refers to any of the mammalianclass I tyrosine kinase receptors. Examples of such receptors includethe ErbB-1 receptor (also known as "the EGF receptor"), the ErbB-2receptor (also called "the HER2 receptor"), the ErbB-3 (or "HER3")receptor, and the ErbB-4 (or "HER4") receptor. The phrase "capable ofbinding" is used to describe a polypeptide that binds anotherpolypeptide with a dissociation constant (K_(d)) of at least 1 mM.

Exemplary heregulin variants that are capable of binding the ErbB-3 andErbB-4 receptors are discussed above and in the examples. The productionof additional heregulin variants, having further modifications (e.g,additional amino acid substitutions, additions, insertions, ordeletions, or covalent modifications) and of chimeric heregulin variantsis within the level of skill in the art.

Furthermore, in light of the teachings herein, those skilled in the artcan design a large number of additional variants that preserve thebinding activity of the heregulin variants of the invention. Forexample, a conservative substitution in a noncritical residue of aheregulin variant (as identified in Example 2) is not expected tosignificantly alter ErbB receptor binding. Moreover, any effects on ErbBreceptor binding can readily be determined in a simple binding assay,such as those described in Examples 1-3. Thus, the invention encompassesall heregulin variants having amino acid substitutions at the specificpositions discussed above, regardless of any additional modificationsthat may be present.

In addition to ErbB receptor binding, a heregulin variant of theinvention can possess one or more other biological activities of anative heregulin. For example, the heregulin variant can also have theability to activate an ErbB receptor. The phrase "ability to activate anErbB receptor" refers to the ability to cause the intracellular kinasedomain of an ErbB receptor to phosphorylate tyrosine residues.Generally, receptor activation involves binding of a heregulin to areceptor complex of two or more ErbB receptors (e.g., an ErbB-2/ErbB-3or ErbB-2/ErbB-4 complex). Receptor binding activates a kinase domain ofone or more of the receptors, which results in phosphorylation oftyrosine residues in one or more of the receptors and/or phosphorylationof tyrosine residues in additional substrate polypeptides(s). ErbBreceptor phosphorylation can be quantified using the tyrosinephosphorylation assays described in Example 3.

Furthermore, a heregulin variant of the invention can be capable ofenhancing the survival, proliferation, and or differentiation of cellshaving suitable ErbB receptors. The phrase "enhancing survival of cells"refers to increasing the period of existence of cells, either in vitroor in vivo, relative to the period of existence of cells that have notbeen exposed to the heregulin variant ("untreated cells").

The expression "enhancing proliferation of cells" means increasing therate or number of mitotic divisions, either in vitro or in vivo,relative to untreated cells. An increase in cell proliferation in cellculture can be detected by counting the number of cells before and afterexposure to the heregulin variant or by microscopic examination of thedegree of confluency. Cell proliferation can also be quantified bymeasuring ³ H-thymidine uptake by the cells.

The phrase "enhancing differentiation of cells" refers to increasing theextent of cell specialization. Cell specialization is characterized bythe acquisition of one or more characteristics that differ from those ofthe original cells. Thus, the extent of cell specialization is typicallydetermined by screening for a change in the phenotype of the cell (e.g.,identifying a change in cellular morphology).

Exemplary cells that express ErbB receptors, and are thereforeresponsive to heregulins, include SK-BR-3 cells, glial cells,glioblastoma cells, Schwann cells, hepatocytes, epithelial cells, andmuscle cells. Glial cells are derived from the central nervous systemand include oligodendrocytes and astrocytes. Muscle cells expressingErbB receptors include muscle cell precursors (myoblasts) as well as themore specialized skeletal, cardiac, and smooth muscle cells.

Other biological activities that a heregulin variant of the inventioncan possess include induction of ion channel (e.g. Na⁺ channel)formation; induction of acetylcholine receptor synthesis at theneuromuscular junction; enhancement of the formation of a synapticjunction between a neuron and a muscle, nerve, or glandular cell;downregulation of estrogen receptor; and cell internalization (possiblyassociated with nuclear localization).

A heregulin variant is produced by any suitable method, includingpeptide synthesis and recombinant techniques. Generally, recombinanttechniques, which are described in detail below, are employed for aheregulin variant longer than about 50 or amino acids.

A heregulin variant having enhanced specificity for the ErbB-4 receptor,relative the ErbB-3 receptor, can be prepared by mutagenizing at one ormore heregulin residues and selecting variants having enhanced ErbB-4receptor specificity. Generally, a residue chosen for mutagenesis inthis context is characterized by a difference in effect on binding tothe ErbB-4 receptor, as compared the effect on binding to the ErbB-3receptor. Such residues are evident, for instance, from thealanine-scanning data presented in Example 2.

Variants having enhanced ErbB-4 receptor specificity are selected byscreening for binding to ErbB-4 receptor using any suitable screeningmethod, such as monovalent phage display, which described in Example 4.The results can be improved by subjecting variants to"counter-selection," which in this case entails the removal of variantsthat bind with high affinity to the ErbB-3 receptor. Example 4demonstrates that counter-selection against ErbB-3-Ig produces asignificant enrichment in variants exhibiting greater specificity forErbB-4-Ig, relative to ErbB-3-Ig, than that of wild-type HRG-β1.

NUCLEIC ACID MOLECULES

The present invention also includes a nucleic acid molecule related tothe heregulin variant. The term "nucleic acid molecule" encompassessingle-stranded and double-stranded DNA molecules, including genomicDNA, cDNA, DNA produced by an amplification reaction (such as polymerasechain reaction ["PCR"]), and DNA produced by oligonucleotide synthesis,as well as RNA molecules, such as mRNA. Genomic DNA can includenon-transcribed and transcribed regions (such as 5' and 3' non-codingregions, introns, and heregulin variant coding regions). cDNA and mRNAmolecules contain sequences corresponding to transcribed regions.

A nucleic acid molecule according to the invention has a nucleotidesequence not found in nature and encodes, or is complementary to anucleic acid molecule encoding, a heregulin variant of the invention ora fragment thereof. A complementary nucleotide sequence is capable offorming Watson-Crick bonds with its complement, in which adenine pairswith thymine or uracil and guanine pairs with cytosine. Adouble-stranded DNA molecule encodes one of the heregulin variants,whereas a single-stranded DNA or RNA molecule is either the coding(sense) strand or the noncoding (anti-sense) strand. When the nucleicacid molecule encodes (or is complementary to a nucleic acid moleculeencoding) a fragment of a heregulin variant, the fragment includes atleast one amino acid substitution.

Because of the redundancy of the genetic code, there are a large numberof possible nucleic acid molecules related to each heregulin variant.More specifically, because several different codons encode the sameamino acid, a large number of different nucleic acid molecules encode(or are complementary to a nucleic acid molecule encoding) the sameheregulin variant.

Generally, a heregulin variant of the invention is produced by mutatinga naturally occurring DNA sequence to introduce the desired mutationsinto the heregulin variant amino acid sequence. However, it may also beadvantageous to change one or more codons in a nucleic acid moleculewithout altering the encoded amino acid. Examples of such "silentmutations" within the scope of the present invention include, forexample, mutations that create or destroy restriction endonuclease sitesto facilitate construction of a desired vector and mutations thatenhance expression of the encoded heregulin variant. Examples of thelatter include nucleotide substitutions designed to reduce the formationof 5' stem and loop structures in the transcribed mRNA or to providecodons that are more readily transcribed by the selected host (e.g., thewell-known preference codons for E. coli or yeast expression).

A nucleic acid molecule of the invention can be incorporated into avector (as described further below) or used, for example, as ahybridization probe or an amplification primer. A hybridization probeaccording to the present invention is useful for detecting a nucleicacid molecule containing a desired mutation, such as, for example, inscreening bacterial transformants to identify clones containing themutated nucleic acid molecule.

Such probes are generally at least about 20 nucleotides and usually lessthan two kilobases. The probe includes a number of nucleotides that issufficient, under the hybridization conditions used, to hybridize with amutated sequence to be detected and to be substantially free fromhybridization with other sequences. Typically, a probe of the presentinvention is at least about 50 nucleotides, and usually about 100nucleotides in length.

An amplification primer according to the invention can be used in aconventional amplification protocol, such as PCR, to detect a nucleicacid molecule containing a desired mutation or to produce sufficientamounts of such a molecule for sequencing, insertion into a vector, etc.An amplification primer is typically used as a member of a primer pair,including a 5' upstream primer that hybridizes with the 5' end of thenucleic acid sequence to be amplified and a 3' downstream primer thathybridizes with the complement of the 3' end of the sequence to beamplified.

In general, a primer according to the invention includes a number ofnucleotides that is sufficient, under the hybridization conditions used,to hybridize with a mutated sequence and to be substantially free fromhybridization with other sequences. The specificity of the primerincreases with the number of nucleotides that hybridize with the mutatedsequence. In addition, specificity is correlated with the proportion ofresidues in the primer that hybridize with the mutated sequence. Aprimer of the present invention generally includes at least about 15nucleotides, and usually at least about 20 nucleotides. The primer neednot exceed about 30 nucleotides, and usually does not exceed about 25nucleotides. In one variation of this embodiment, the primer includesbetween about 20 and about 25 nucleotides. Generally, the primers shouldhave a T_(m) in the range of about 55° C. to about 75° C. In practice,the T_(m) is usually between about 60° C. to about 65° C. to facilitateamplification under stringent conditions.

VECTORS AND HOST CELLS

A nucleic acid molecule of the present invention can be incorporatedinto a vector for propagation and/or expression in a host cell. Suchvectors typically contain a replication sequence capable of effectingreplication of the vector in a suitable host cell (i.e., an origin ofreplication) as well as sequences encoding a selectable marker, such asan antibiotic resistance gene. Upon transformation of a suitable host,the vector can replicate and function independently of the host genomeor integrate into the host genome. Vector design depends, among otherthings, on the intended use and host cell for the vector, and the designof a heregulin variant vector for a particular use and host cell iswithin the level of skill in the art.

If the vector is intended for expression of a heregulin variant, thevector includes one or more control sequences capable of effectingand/or enhancing the expression of an operably linked heregulin variantcoding sequence. Control sequences that are suitable for expression inprokaryotes, for example, include a promoter sequence, an operatorsequence, and a ribosome binding site. Control sequences for expressionin eukaryotic cells include a promoter, an enhancer, and a transcriptiontermination sequence (i.e., a polyadenylation signal).

The term "operably linked" means that two nucleic acid sequences are ina functional relationship with one another. For example, a promoter (orenhancer) is operably linked to a coding sequence if it effects (orenhances) the transcription of the sequence. A ribosome binding site isoperably linked to a coding sequence if it is positioned to facilitatetranslation. Operably linked nucleic acid sequences are oftencontiguous, but this is not a requirement. For example, enhancers neednot be contiguous with a coding sequence to enhance transcription of thecoding sequence.

A heregulin variant expression vector can also include other sequences,such as, for example, nucleic acid sequences encoding a signal sequenceor an amplifiable gene. As discussed above, a signal sequence directsthe secretion of a polypeptide fused thereto from a cell expressing thechimeric protein. In the expression vector, nucleic acid encoding asignal sequence is linked to a heregulin variant coding sequence so asto preserve the reading frame of the heregulin variant coding sequence.The inclusion of an amplifiable gene (e.g., the dihydrofolate reductase[DHFR] gene) in a heregulin variant expression vector allows selectionof host cells containing multiple copies of the nucleic acid moleculeencoding the heregulin variant.

A vector of the present invention is produced by linking desiredelements by ligation at convenient restriction sites. If such sites donot exist, suitable sites can be introduced by standard mutagenesis(e.g., site-directed or cassette mutagenesis) or syntheticoligonucleotide adaptors or linkers can be used in accordance withconventional practice.

The present invention also provides a host cell containing a vector ofthis invention. A wide variety of host cells are available forpropagation and/or expression of vectors. Examples include prokayoticcells (such as E. coli and strains of Bacillus, Pseudomonas, and otherbacteria), yeast or other fungal cells, insect cells, plant cells, andphage, as well as higher eukaryotic cells (such as Chinese hamster ovarycells and other mammalian cells). Host cells according to the inventioninclude cells in culture and cells present in live animals, such astransgenic animals. See U.S. Pat. No. 5,364,934 for more information onvectors and host cells suitable for use in the recombinant production ofa heregulin variant.

A vector of the present invention is introduced into a host cell by anyconvenient method, which will vary depending on the vector-host systememployed. Generally, a vector is introduced into a host cell bytransformation (also known as "transfection") or infection with a virus(e.g., phage) bearing the vector. If the host cell is a prokaryotic cell(or other cell having a cell wall), convenient transformation methodsinclude the calcium treatment method described by Cohen et al., PNAS USA69:2110-14 (1972), and the polyethylene glycol method of Chung et al.,Nuc. Acids. Res. 16:3580 (1988). If a prokaryotic cell is used as thehost and the vector is a phagemid vector, the vector can be introducedinto the host cell by infection, as described in Example 1. Yeast cellscan be transformed using polyethylene glycol, for example, as taught byHinnen, PNAS U.S.A. 75:1929-33 (1978). Mammalian cells are convenientlytransformed using the calcium phosphate precipitation method describedby Graham et al., Virology 52:546 (1978), and Gorman et al., DNA andProtein Eng. Tech. 2:3-10 (1990). However, other known methods forintroducing DNA into host cells, such as nuclear injection,electroporation (see Example 1), and protoplast fusion also are suitablefor use in the invention.

In one embodiment, a host cell containing a nucleic acid moleculeencoding a heregulin variant is produced by homologous recombination, asdescribed in WO 91/06667. Briefly, this method involves transforming ahost cell containing an endogenous heregulin gene with a homologousrecombination vector that includes the sequence to be introduced. Thehomologous recombination vector also includes at least one sequence ofat least about 150 nucleotides in length that is homologous with anendogenous sequence flanking the endogenous heregulin gene. Suitableflanking sequences are readily identified, for example, by the method ofgenomic walking, using a known native heregulin nucleic acid sequence asa starting point. The homologous recombination vector additionallyincludes an amplifiable gene, such as the DHFR gene.

Transformation is carried out under conditions such that the vectorintegrates into the host cell genome by recombination. Cells thatintegrate the vector are then cultured under conditions that select foramplification of the amplifiable gene. The resulting cells are thenscreened for high levels of heregulin variant production.

RECOMBINANT PRODUCTION OF HEREGULIN VARIANTS

To produce a heregulin variant recombinantly, host cells containing aheregulin variant expression vector are prepared and cultured underconditions suitable for cell growth and for expression of the heregulinvariant. In particular, the culture medium contains appropriatenutrients and growth factors for the host cell employed. The nutrientsand growth factors required for growth of a selected host cell are, inmany instances, well known or can be readily determined empirically bythose skilled in the art. Suitable culture conditions for mammalian hostcells, for instance, are described in Mammalian Cell Culture (Mathered., Plenum Press 1984), and in Barnes and Sato, Cell 22:649 (1980).

In addition, the culture conditions should allow transcription,translation, and protein transport between cellular compartments.Factors that affect these processes are well-known and include, forexample, DNA/RNA copy number; factors that stabilize RNA; nutrients,supplements, and transcriptional inducers or repressors present in theculture medium; temperature,pH, and osmolality of the culture; and celldensity. The adjustment of these factors to promote expression in aparticular vector-host cell system is within the level of skill in theart. Principles and practical techniques for maximizing the productivityof in vitro mammalian cell cultures, for example, can be found inMammalian Cell Biotechnology: a Practical Approach (Butler ed., IRLPress 1991).

The cell culture procedure employed in the production of a heregulinvariant of the present invention can be any of a number of well-knownprocedures for large- or small-scale production of proteins. Theseinclude, but are not limited to, the use of a fluidized bed bioreactor,a hollow fiber bioreactor, a roller bottle culture system, and a stirredtank bioreactor system. A heregulin variant can be produced, forinstance, in a batch, fed-batch, or continuous mode process.

Methods for recovery of recombinant proteins produced as described aboveare well-known and vary depending on the expression system employed. Forexample, if, as is typical, the heregulin variant is fused to a signalsequence, the heregulin variant is recovered from the culture medium orthe periplasm. Conveniently, the variant is secreted into theperiplasmic space as a mature protein. The heregulin variant can also beexpressed intracellularly and recovered from cell lysates.

The heregulin variant can be purified from culture medium or a celllysate by any method capable of separating the variant from componentsof the host cell or culture medium. Typically the heregulin variant isseparated from host cell and/or culture medium components that wouldinterfere with the intended use of the heregulin variant. As a firststep, the culture medium or cell lysate is usually centrifuged orfiltered to remove cellular debris. The supernatant is then typicallyconcentrated or diluted to a desired volume or diafiltered into asuitable buffer to condition the preparation for further purification.

The heregulin variant is typically further purified in the same manneras the most homologous native heregulin, taking account of anysubstantial differences in properties between the two molecules. Forexample, if the heregulin variant is an epitope-tagged heregulinvariant, purification can be carried out using an immunoaffinity columncontaining antibody to the epitope tag. The following exemplaryprocedures for purifying heregulins can be used or adapted for purifyinga heregulin variant of the invention: fractionation on an immunoaffinitycolumn, fractionation on an ion-exchange column, ammonium sulphate orethanol precipitation, reverse phase HPLC, chromatography on silica,chromatography on heparin Sepharose, chromatography on a cation exchangeresin, chromatofocusing, SDS-PAGE, and gel filtration (e.g., using aHigh Load Superdex 75™ prep grade column).

If the heregulin variant is expressed initially as an insoluble,aggregated form (especially in bacterial host cells), it may benecessary to solubilize and renature the heregulin variant usingtechniques available in the art for solubilizing and renaturingrecombinant protein refractile bodies. See, e.g., U.S. Pat. No.4,511,502.

In one variation of this embodiment, the heregulin variant is purified(1) to a degree sufficient to obtain at least 15 residues, andpreferably 20 residues, of N-terminal or internal amino acid sequence,using a spinning cup sequenator, or (2) to homogeneity by SDS-PAGE undernon-reducing or reducing conditions using Coomassie blue stain. As usedherein, "homogeneity" means less than about 5% contamination with othersource proteins, as determined by staining with Coomassie blue.

UTILITY OF HEREGULIN VARIANTS

Generally speaking, heregulin variants according to the invention can beused in the same applications as native heregulins. Of course, someheregulin variants within the scope of the invention may be bettersuited for one application than for other applications. However, thoseskilled in the art can readily ascertain which heregulin variants areappropriate for a given application by using one or more conventionalassays to determine the biological activity of the variants.

Pharmaceutical Compositions and Treatment Methods

Heregulins are useful in treating a wide range of diseases and disordersaffecting the nervous system, musculature, and epithelia. In addition,heregulins can be used in the treatment of cancer. As used herein,"treatment" encompasses the treatment of an existing disease or disorderas well as prophylactic measures.

Accordingly, the present invention provides a pharmaceutical compositionincluding a heregulin variant that is useful in treating any of avariety of diseases or disorders. In one embodiment, a pharmaceuticalheregulin variant composition is employed to treat a mammal. Inparticular, the composition is useful for treating humans, farm animals(e.g., cows and sheep), zoo animals, animals used in sports (e.g.,horses), and pets (e.g., dogs and cats). In a variation of thisembodiment, the composition is used to treat a human patient.

A heregulin variant according to the invention can be useful inpromoting the development, maintenance, and/or regeneration of a neuronin vivo. Neurons that respond to such a variant include central nervoussystem (brain and spinal chord) neurons, peripheral nervous systemneurons (including sympathetic, parasympathetic, sensory, and entericneurons), and motorneurons. Diseases or disorders amenable to heregulinvariant treatment arise in individuals who have suffered nervous systemdamage due, for example, to trauma, surgery, stroke, ischemia,infection, metabolic disease, nutritional deficiency, malignancy, or atoxic agent.

A heregulin variant can provide therapeutic benefits to such individualsby promoting the survival, proliferation, or differentiation of neurons.For example, a heregulin variant can be used to promote the survival orproliferation of motorneurons that have been damaged by trauma orsurgery. A heregulin variant can also be employed to treat motoneurondisorders, such as amyotrophic lateral sclerosis (Lou Gehrig's disease),Bell's palsy, and various conditions involving spinal muscular atrophyor paralysis. In addition, a heregulin variant can also be useful fortreating a human neurodegenerative disorder, such as Alzheimer'sdisease, Parkinson's disease, epilepsy, multiple sclerosis, Huntington'schorea, Down's Syndrome, nerve deafness, and Meniere's disease.

Furthermore, a heregulin variant of the invention can be used to treatneuropathy, especially peripheral neuropathy. As used herein, the term"peripheral neuropathy" refers to a disorder affecting the peripheralnervous system, most often manifested as one or a combination of motor,sensory, sensorimotor, or autonomic neural dysfunctions. Examplesinclude, but are not limited to, distal sensorimotor neuropathy andautonomic neuropathies, such as reduced motility of the gastrointestinaltract or atony of the urinary bladder. Peripheral neuropathies amendableto heregulin variant treatment can be inherited, can result from asystemic disease, or can be induced by a toxic agent. Examples ofhereditary neuropathies include Charcot-Marie-Tooth disease, Refsum'sdisease, Abetalipoproteinemia, Tangier disease, Krabbe's disease,Metachromatic leukodystrophy, Fabry's disease, and Dejerine-Sottassyndrome. Examples of neuropathies associated with systemic diseaseinclude post-polio syndrome; and examples of neuropathies induced by atoxic agent include those caused by treatment with a chemotherapeuticagent.

A heregulin variant can also be employed to improve neural function. Thebeneficial effects of heregulin variant treatment are attributed toinduction of the formation of ion channels in cell membranes andenhancement of the formation of synaptic junctions.

A heregulin variant according to the invention can also be used to treatmuscle cells and medical conditions affecting muscle cells. Inparticular, such heregulin variant can be useful for treating muscledamage, decreasing atrophy of muscle cells, and increasing muscle cellsurvival, proliferation and/or regeneration. Examples ofpathophysiological conditions of the musculature amenable to treatmentwith a heregulin variant include skeletal muscle diseases (e.g.,myopathy or dystrophy), cardiac muscle disorders (including atrialcardiac arrhythmias, cardiomyopathy, ischemic damage, congenitaldisease, and cardiac trauma), and smooth muscle disorders (such asarterial sclerosis, vascular lesion, or congenital vascular disease). Aheregulin variant can also be employed to reduce hypertension and toincrease functional acetylcholine receptors on muscle cells (e.g., inindividuals having myasthenia gravis or tachycardia).

A heregulin variant of the invention can also enhance repair and/orregeneration of tissues that express ErbB receptors, especially ErbB-2and either ErbB-3 or ErbB-4 receptors. Accordingly, a heregulin variantcan be useful for treating dermal wounds, gastrointestinal disease,Barrett's esophagus, cystic or non-cystic end stage kidney disease, orinflammatory bowel disease. A heregulin variant can also be employed topromote reepithelialization in the human gastrointestinal, respiratory,reproductive, or urinary tract.

In addition, a heregulin variant according to the invention can beuseful for inhibiting tumor cell invasion and metastasis. In particular,a tumor characterized by reduced endogenous heregulin levels (Park etal., Proc. Am. Assoc. Cancer Res. 34:521 [1993]) is responsive totreatment with a heregulin variant of the invention. Additionally, atumor that overexpresses ErbB receptors can be treated by using aheregulin variant conjugated to a cytotoxic agent (described above) todirect the cytotoxic agent to the tumor tissue. A heregulinvariant-enzyme conjugate can also be employed to target a prodrug(described above) therapy to cells expressing ErbB receptors.

A pharmaceutical composition according to the invention is prepared forstorage by mixing a heregulin variant having the desired degree ofpurity with an optional physiologically acceptable carrier, excipient,or stabilizer, such as are described in Remington's PharmaceuticalSciences 16th Edition (Osol ed., 1980). The composition can be stored inthe form of a lyophilized cake or an aqueous solution. Apharmaceutically acceptable carrier, excipient, or stabilizer isnon-toxic to recipients at the dosages employed, and can include abuffer (such as a phosphate buffer, citrate buffer, and buffers madefrom other organic acids), an antioxidant (e.g., ascorbic acid), alow-molecular weight (less than about 10 residues) polypeptide, aprotein (such as serum albumin, gelatin, and an immunoglobulin), ahydrophilic polymer (such as polyvinylpyrrolidone), an amino acid (suchas glycine, glutamine, asparagine, arginine, and lysine), amonosaccharide, a disaccharide, and other carbohydrates (includingglucose, mannose, and dextrins), a chelating agent (e.g.,ethylenediaminetetratacetic acid [EDTA]), a sugar alcohol (such asmannitol and sorbitol), a salt-forming counterion (e.g., sodium), and/ora nonionic surfactant (such as Tween™, Pluronics™, and PEG). In oneembodiment, the physiologically acceptable carrier is an aqueouspH-buffered solution.

A heregulin variant composition intended for in vivo administration istypically sterile. Sterilization is readily accomplished by filtrationthrough a sterile filtration membrane. If the composition is stored inlyophilized form, the composition can be filtered before or afterlyophilization and reconstitution.

A pharmaceutical heregulin variant composition of the invention isgenerally placed into a container having a sterile access port, such as,for example, an intravenous solution bag or a vial having a stopperpierceable by a hypodermic injection needle.

Methods for administering a pharmaceutical heregulin variant compositiondo not differ from known methods for administering therapeutic proteins.Suitable routes of administration include, for example, intravenous,intraperitoneal, intracerebral, intramuscular, intraocular,intraarterial, or intralesional routes. A pharmaceutical heregulinvariant composition can be administered continuously by infusion or bybolus injection.

If desired, a sustained-release preparation can also be used toadminister a heregulin variant. An exemplary sustained-releasepreparation has a semipermeable matrix of a solid hydrophobic polymer towhich the heregulin variant is attached. Examples of suitable polymersinclude a polyester, a hydrogel, a polylactide, a copolymer ofL-glutamic acid and γ-ethyl-L-glutamate, non-degradable ethylene-vinylacetate, a degradable lactic acid-glycolic acid copolymer, andpoly-D-(-)-3-hydroxybutyric acid. Such matrices are in the form ofshaped articles, such as films, or microcapsules.

In one embodiment, a sustained-release heregulin variant preparationincludes a liposomally entrapped heregulin variant. Liposomes are smallvesicles composed of various types of lipids, phospholipids, and/orsurfactants. These components are typically arranged in a bilayerformation, similar to the lipid arrangement of biological membranes.Liposomes containing heregulin variants are prepared by known methods,such as, for example, those described in Epstein et al., PNAS USA82:3688-92 (1985), and Hwang et al., PNAS USA 77:4030-34 (1980).Ordinarily the liposomes in such preparations are of the small (about200-800 Angstroms) unilamellar type in which the lipid content isgreater than about 30 mol. percent cholesterol, the specific percentagebeing adjusted to provide the optimal therapy. Useful liposomes can begenerated by the reverse-phase evaporation method, using a lipidcomposition including, for example, phosphatidylcholine, cholesterol,and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes areextruded through filters of defined pore size to yield liposomes withthe desired diameter.

For treatment of neurologic diseases or disorders, a heregulin variantcan be adsorbed onto a membrane, such as a silastic membrane, which canbe implanted in proximity to damaged neural tissue, as described in WO91/04014.

The dosage of a heregulin variant composition to be employedtherapeutically depends, for example, upon the therapeutic objectives,the route of administration, and the condition of the patient.Accordingly, it is necessary for the clinician to titer the dosage andmodify the route of administration as required to obtain the optimaltherapeutic effect. A typical daily dosage can range from about 1 μg/kgto up to 100 mg/kg of body weight or more per day, but is typicallybetween about 10 μg/kg/day to 10 mg/kg/day.

Generally, the clinician begins with a low dosage of a pharmaceuticalheregulin variant composition and increases the dosage until the desiredtherapeutic effect is achieved.

The administration of a heregulin variant of the invention can becombined with other therapeutic regimens. For the treatment ofneurological conditions, a heregulin variant is optionally combined, oradministered in concert, with another neurotrophic factor to achieve adesired therapeutic effect. For example, a heregulin variant can be usedtogether with nerve growth factor (NGF), a neurotrophin (e.g., NT-3, -4,or -5), bone-derived nerve factor (BDNF), an insulin-like growth factor(e.g., IGF-1 or IGF-2), gas6, or another neurotrophic factor to achievea synergistic stimulatory effect on neurons. Suitable dosages for theneurotrophic factors do not differ from those known in the art for suchmolecules.

For the treatment of cancer, radiation and/or a chemotherapeutic agentcan be administered concomitantly with a heregulin variant. Suitablepreparation and dosing schedules for such chemotherapeutic agents are asrecommended by the manufacturer or as determined empirically by theclinician. For preparation and dosing schedules for standardchemotherapeutic agents, see Chemotherapy Service (Perry ed., Williams &Wilkins 1992). Administration of the chemotherapeutic agent can precede,or follow, administration of the heregulin variant, or thechemotherapeutic agent can be given simultaneously therewith. Antibodiesagainst tumor-associated antigens, such as antibodies that bind EGFR,ErbB-2, ErbB-3, or ErbB-4 receptor, or vascular endothelial factor(VEGF) can also be co-administered with an heregulin variant, as can oneor more cytokines.

Non-Therapeutic Methods

Heregulin variants according to the invention also be employed in avariety of non-therapeutic applications, such as cell culture methodsand diagnostic methods. For example, a heregulin variant can be used topromote the ex vivo survival, proliferation, or differentiation ofcells, such as glial, Schwann, and muscle cells. Cultures of such cellsare useful for producing cell-specific factors, such as, for example,the nerve growth factor receptor (P75^(NGFR)) which is a Schwanncell-specific factor. Cell-specific factors can be employed directly asdiagnostic tools or employed to generate antibodies for diagnostic use.

Ex vivo cell cultures can also be used as cellular prostheses fortransplantation. For example, Schwann cell cultures can be transplantedinto areas of damaged spinal cord to promote regeneration of interruptedcentral axons or can be used to assist the repair of peripheral nerveinjuries.

Accordingly, the present invention provides a cell culture method inwhich heregulin-responsive cells are provided in a suitable cell culturemedium. Suitable tissue culture media are well known to those skilled inthe art and include, but are not limited to, Minimal Essential Medium(MEM), RPMI-1640, and Dulbecco's Modified Eagle's Medium (DMEM). Thesetissue culture media are commercially available from Sigma ChemicalCompany (St. Louis, Mo.) and GIBCO (Grand Island, N.Y.). The cells arecultured in the cell culture medium under conditions that permit thecells to grow in the presence of a heregulin variant. Suitableprocedures for cell culture do not differ from known procedures andinclude, for example, liquid culture, culture in agar, and culture in aclot.

The cells are cultured in the presence of an effective amount of aheregulin variant. The amount of heregulin variant can vary, dependingon the cell type and cell culture conditions, but generally is in therange of about 10 ng/ml to about 1 mg/ml. An appropriate concentrationfor a given cell culture can readily be determined empirically by thoseskilled in the art.

Techniques for culturing Schwann cells ex vivo are described in Li etal. (supra), and Sklar et al. (supra) describe ex vivo culture of clonalhuman myoblasts. A heregulin variant of the invention can replace theother heregulin polypeptides used in these methods.

A heregulin variant can also be employed in the diagnosis of a cancercharacterized by erbB (e.g., erbB2) overexpression. In a diagnosticmethod according to the invention, a sample is obtained from anindividual and contacted with a heregulin variant under conditions thatallow specific binding between the variant and any ErbB receptorspresent in the sample. The sample can be a tissue sample, a bodily fluidsample, or a cell. In the case of a solid tumor, a tissue sample can betaken from a surgically removed tumor and prepared for testing byconventional techniques. In the case of lymphomas and leukemias, asample containing lymphocytes, leukemic cells, or lymph tissues isobtained. Other samples, including samples of urine, tear drops, serum,cerebrospinal fluid, feces, sputum, cell extract, and the like, can beuseful for diagnosing particular tumors. As used with regard to thismethod, the term "specific binding" means that the heregulin variantbinds an ErbB receptor with an affinity that is sufficiently high thatthe heregulin variant does not substantially cross-react with othercomponents present in the sample under the suitable reaction conditions.

The amount of heregulin variant that specifically binds to the sample isdetermined as an indication of ErbB receptor content. For example, atissue sample can be obtained from a primary tumor and used to prepareformalin-fixed, paraffin-embedded blocks. See Muss et al., supra; Presset al., Cancer Research 54:2771-77 (1994). Tissue sections are thenprepared according to known techniques.

A heregulin variant is contacted with a tissue section under conditionsthat permit specific binding between the variant and ErbB receptorspresent in the section. Binding is generally detected using a label,such as a radioisotope, a fluorescent label, or an enzyme-substratelabelling system. The label can be conjugated directly to the heregulinvariant, as described above.

Alternatively, the label can be bound to the heregulin variantindirectly. For example, the label can be conjugated to ananti-heregulin variant antibody or conjugated to biotin or avidin andused with an anti-heregulin variant antibody conjugated to avidin orbiotin (respectively), as described generally in Current Protocols inImmunology (supra). Selective binding between biotin and avidin linksthe label to the heregulin variant.

While in vitro analysis is normally contemplated, in vivo analysis usinga heregulin variant conjugated to a suitable detectable label (e.g., Infor imaging) can also be performed. See, e.g., U.S. Pat. No. 4,938,948.

A diagnostic method of the invention can be used in combination withother diagnostic/prognostic evaluations such as determining lymph nodestatus, primary tumor size, histologic grade, estrogen or progesteronestatus, tumor DNA content (ploidy), or cell proliferation (S-phrasefraction). See Muss et al., New Eng. J. Med. 330:1260-66 (1994).

A heregulin variant according to the invention is also useful as astandard in assays for heregulins (such as a radioimmunoassay, anenzyme-linked immunoassay, and a radioreceptor assay), in an affinitypurification technique (e.g., for an ErbB receptor such as ErbB-3 orErbB-4 receptor), and in a competitive receptor binding assay. Aheregulin variant can also be employed as an immunogen for generatinganti-heregulin variant antibodies useful in detection and/orpurification of heregulin variants.

In addition to the applications discussed above, a heregulin variantthat has an enhanced specificity for the ErbB-4 receptor, relative tothe ErbB-3 receptor, can be used to bind and stimulate the ErbB-4receptor preferentially over the ErbB-3 receptor. Such a variant isuseful in studies designed to distinguish between the presence of ErbB-3and ErbB-4 receptors in a sample or to determine whether particularbiological actions are mediated by one or both receptors. For instance,in cell types having a relatively small number of ErbB-4 receptors and alarge number of ErbB-3 receptors, in the absence of receptor-specificheregulin variants, it is difficult to identify signal transductionpathways activated by the different receptors and to link particularbiological actions to one or both receptors. A variant thatpreferentially binds to the ErbB-4 receptor can be used to helpelucidate the roles of the ErbB-3 and ErbB-4 receptors in heregulinaction.

Diagnostic Kits and Articles of Manufacture

The present invention also provides kits useful in practicing theabove-described methods. In one embodiment, the invention provides adiagnostic kit, i.e., a packaged combination of reagents for use intesting a sample. The components of the kit are typically provided inpredetermined ratios. A kit for detecting ErbB receptors can include,for example, a heregulin variant labelled with a suitable label or aheregulin variant with a labelled reagent(s) for indirect labelling. Ifthe label is an enzyme, the kit typically includes any substrate orcofactor required by the enzyme label. Other additives, such asstabilizers, buffers and the like, can also be included in the kit. Kitreagents can be provided as dry powders, usually lyophilized, togetherwith excipients for preparing kit reagent solutions of the appropriateconcentration. Kits also typically include instructions for carrying outthe assay for which the kit is designed.

The present invention also provides an article of manufacture containinga pharmaceutical heregulin variant composition useful for the treatmentof a disorder described above. The article of manufacture includes acontainer and a label. Suitable containers include, for example,bottles, vials, syringes, and test tubes. The container can be made fromany of a variety of materials, such as glass or plastic and can have asterile access port. The label on, or associated with, the containerindicates the disorder that the composition is to be used to treat.

The article of manufacture can be a component of a kit that includes asecond container including a pharmaceutically-acceptable buffer, such asphosphate-buffered saline, Ringer's solution, or dextrose solution. Thekit can also include other materials that are desirable from acommercial or user standpoint, such as other buffers, diluents, filters,needles, syringes, and package inserts with instructions for use.

The following examples are offered by way of illustration and not by wayof limitation. The disclosures of all citations in the specification areexpressly incorporated herein by reference.

EXAMPLE 1 Determination of the Heregulin-β1 Minimal EGF-Like Domain

The smallest portion of HRG-β1 EGF that provides high-affinity receptorbinding in the context of phage display was determined by preparingphagemid vectors that produced HRG-β1 147-227, 147-244, 177-227, or177-244 fused to the C-terminus of M13 pIII. These HRG-β1 EGF-likedomain fragments were amplified from the vector pHL89 (which isdescribed in Holmes, et al., Science 256: 1205-10 [1992]) by PCR withprimers having NsiI/XbaI-containing overhangs.

These fragments were inserted into the phagemid display vector pam-g3 byrestriction digest-ligation at the same sites to generate constructspHRG1-g3 (177-227), pHRG2-g3 (177-244), pHRG4-g3 (147-227), and pHRG5-g3(147-244). pam-g3 was a derivative of phGHam-g3, which was designed forphage display of human growth hormone (hGH) and was described in Lowmanet al., Biochemistry 30:10832-38 (1991). pam-g3 was produced by removingthe hGH gene present in phGHam-g3 and replacing this gene with a stufferfragment, which provides space for cleavage at the restriction sitesused for cloning.

pHRG1-g3 contained an Ala227Val mutation that had been introduced ingenerating the construct. Single-stranded uracil-containing template wasproduced from this construct and used in site-directed mutagenesis torestore Ala227, generating pHRG6-g3 (177-227). In each of theabove-described constructs, the HRG-β1 EGF-like domain fragment wasattached to residue 247 of pIII.

In an effort to determine whether the inclusion of an extended flexiblelinker at the junction between HRG-β1 EGF and pIII would alleviatepotential pIII interference of binding to ErbB-3, two constructs havinglinkers at this junction were prepared from the pHRG1-g3 template.pHRG8-g3 expressed HRG-β1 177-228 attached to pIII 323 through a linkercontaining three consecutive GGGS (SEQ ID NO:34) repeats, and pHRG11-g3expressed HRG-β1 177-230 attached to pIII 247 through a GGGSGGG (SEQ IDNO:35) linkage.

The HRG-β1 EGF-like domains expressed from the above-describedconstructs are designated herein by removing the "p" and the "-g3" thatappear in the name of the construct. Thus, the HRG-β1 EGF-like domainexpressed from the pHRG2-g3 construct is designated "HRG2."

The domains were displayed monovalently on phage as pIII fusionproteins, as described by Bass et al., Proteins 8:309-14 (1990), andthen analyzed for their binding to the high-affinity ErbB-2/3receptor-Ig fusion (ErbB-2/3-Ig) using the phage ELISA techniquedescribed by Cunningham et al., EMBO J. 13:2508-15 (1994), with slightmodifications. To produce phage displaying the domains, mutagenesisreaction mixtures were electrotransformed into XL1-Blue™ cells(Stratagene, Inc., La Jolla, Calif.), according to the manufacturer'sprotocol. The transformed cells were then infected with 10¹¹plaque-forming units (pfu) M13K07 helper phage (Promega Corp., Madison,Wis.). Phage stocks (about 10¹⁴ phagemid/mL) were prepared byprecipitating culture broths from the cells after 18-24 hours (h) ofgrowth with 20% PEG(2000)/2.5 M NaCl, according to the method ofSambrook and Maniatis, Molecular Cloning a Laboratory Manual Cold SpringHarbor Press 429 (1989). The phage were resuspended inphosphate-buffered saline (PBS: 0.01 M sodium phosphate, 0.1 M NaCl, pH7.5).

ErbB receptor-Ig fusions where prepared using vectors that expressed theextracellular domain (ECD) of the ErbB-2, ErbB-3, or ErbB-4 receptorfused to a human IgG constant domain. First, a unique MluI site wasengineered into a vector expressing a human IgG heavy chain (pDR2) atthe region encoding the hinge domain of the immunoglobulin. MluI siteswere also engineered into a set of ErbB expression vectors at the regionencoding the ECD/transmembrane junctions of these receptors. Allmutagenesis was performed according to the method of Kunkel et al.,Methods Enzymol. 154:367-82 (1987).

The MluI sites were used to make constructs that expressed the desiredErbB receptor-Ig fusion. The fusion junctions of the various receptorfusions were as follows (in order from the N-terminus to theC-terminus): for ErbB-2, Glu646 of ErbB2 was fused toThr-Arg-Asp-Lys-Thr (TRDKT; SEQ ID NO:36), which was fused to His224 ofVH (H224_(VH)); for ErbB-3, Leu617 of ErbB-3 was fused toTRDKT-H224_(VH) ; for ErbB-4, Gly640 of ErbB-4 was fused toTRDKT-H224_(VH). The ErbB receptor residue numbers are indicatedaccording to the numbering system of Plowman et al. PNAS USA 90:1746-50(1993). The conserved TR sequence was derived from the MluI site, andthe conserved DKT sequence was derived from a linker. The finalexpression constructs had a pRK-type plasmid backbone wherein eukaryoticexpression was driven by a cytomegalovirus (CMV) promoter.

Receptor fusions were expressed from these constructs, purified, andallowed to form disulfide-linked dimers. Homodimeric ErbB-2, ErbB-3 andErbB-4 receptor-Ig fusions were produced by transfecting cells with theconstruct encoding the appropriate receptor fusion. Heterodimericreceptor fusions were generated by co-transfecting two expressionvectors encoding different receptor fusions into the same cells. Theresulting secreted receptor fusions were mixtures of two types ofhomodimers and the expected heterodimer.

To express the receptor fusions, adherent HEK-293 cells (ATCC No.CRL1573) were transfected with the appropriate expression vector(s)using the calcium phosphate precipitation method described by Gorman etal., DNA and Protein Eng. Tech. 2:3-10 (1990). Serum-containing mediumwas replaced with serum-free medium at 15 h post-transfection, and thetransfected cells were cultured serum-free for 5-7 days.

The resulting conditioned medium was harvested and passed throughProtein A columns (1 mL Pharmacia HiTrap™, Piscataway, N.J.). Purifiedreceptor fusions were eluted with 0.1 M citric acid (pH 4.2) into tubescontaining 1 M Tris-HCl (pH 9.0). The eluted proteins were then dialyzedagainst PBS and concentrated using Centri-prep-30™ filters (Amicon,Beverly, Mass.). Glycerol was added to a final concentration of 25%, andthe preparations stored at -20° C. The receptor fusion concentration wasdetermined via a Fc-ELISA.

Microtiter plates (Nunc Maxisorp™ 96-well plates, Inter Med, Denmark)for phage ELISA were prepared as follows. The wells were precoatedovernight with 0.5 μg of rabbit anti-human IgG (Fc gammafragment-specific) antibodies (Jackson Immunoresearch, West Grove, Pa.)in 100 μL 50 mM NaCO₃ (pH 9.6). The wells were blocked for 30 minutes(min) with 200 μL PBS containing 0.1% bovine serum albumin (BSA) andrinsed with wash buffer (PBS containing 0.05% Tween 20™). The wells werethen coated with 0.1 μg ErbB-2/3-Ig in binding buffer (PBS, 0.1% BSA,0.05% Tween 20™) for 1 h, and washed again.

Serial dilutions of soluble ErbB-2/3-Ig (competitor) and a concentrationof phage predetermined to give 60% saturation (without competitor) wereadded to the wells in 100 μL binding buffer. Following incubation for 2h at room temperature, plates were washed extensively and treated with a1:900 dilution of anti-M13 horseradish peroxidase conjugate (Pharmacia,Piscataway, N.J.) for 20 min. The amount of phage binding was determinedby assaying horseradish peroxidase activity using o-phenylenediaminedihydrochloride) substrate solution (Sigma Chemical Company, St. Louis,Mo.). EC₅₀ values were calculated as the concentration of solubleErbB-2/3-Ig required to compete half of the phage off the plate.

The results are set forth in Table 3.

                  TABLE 3                                                         ______________________________________                                        Initial Heregulin-Phage Variants                                                          Heregulin-β1                                                                         Linker and pIII                                                                            ErbB-2/3-Ig                                Construct residues fusion point EC.sub.50  (nM)*                            ______________________________________                                        HRG2    177-244     pIII 247       6.0                                          HRG4 147-227 pIII 247 38.0                                                    HRG5 147-244 pIII 247 4.7                                                     HRG6 177-227 pIII 247 40.0                                                    HRG7 177-228 pIII 247 42.0                                                    HRG8 177-228 (1-52) (GGGS).sub.3 -pIII 323 11.0                               HRG11 177-230 GGGSGGG-pIII 247 19.0                                         ______________________________________                                         *Average of duplicate experiments                                        

All phage stocks bound specifically to immobilized ErbB-2/3-Ig and couldbe competed off with similar EC₅₀ values (5-42 nM). These values wereabout 100-fold higher than the previously measured dissociation constant(K_(d)). Holmes, et al., supra. However, EC₅₀ values obtained from phageELISA are often higher than the true K_(d), particularly forhigh-affinity interactions. This may be due to the high receptor coatconcentration required to give a reasonable signal for the bound phage,a low percentage of active receptor in competitor solutions, orinterference from the linkage of the protein to pIII.

In any event, the HRG-β1 147-176 did not appear to enhance the bindingof phage displaying the HRG-β1 EGF-like domain to ErbB-3-Ig, whereasHRG-β1 228-244 contributed slightly to binding affinity. Thus, theminimal EGF-like domain of HRG-β1 was defined as HRG-β1 177-228.

Phage ELISA was also carried out for the constructs encoding HRG-β1EGF-pIII fusions containing linkers inserted between HRG-β1 EGF and thepIII fragment. Mild enhancements in binding affinity were observed forthe linker-containing fusions, along with increased expression offunctional fusions, as determined by binding titrations of the phagestocks. The fusion of HRG-β1 EGF via a linker to pIII residue 323,instead of residue 247 (pHRG8-g3) resulted in a slight affinityenhancement. This construct was therefore used as the template vectorfor construction of phage display libraries.

EXAMPLE 2 Identification of Active Residues in the Heregulin-β1 EGFDomain by Alanine Scanning

This example describes the identification of active residues in theheregulin-β1 (HRG-β1) EGF-like domain (HRG-β1 177-229) that play a rolein the binding of HRG-β1 to the ErbB-3 and ErbB-4 receptors. Activeresidues were identified by mutating individual amino acids in thisdomain to alanine. The mutated domains (hereinafter "variants") weredisplayed monovalently on phage as pIII fusion proteins and variantaffinities for ErbB-3 and ErbB-4 were determined by phage ELISA.Selected variants were expressed as thioredoxin fusion proteins, whichwere also assayed for ErbB-3 and ErbB-4 affinity.

Alanine Scanning Mutagenesis and Phage Display

Alanine-substituted variants were generated by site-directed mutagenesisaccording to Kunkel et al., Methods Enzymol. 154:367-82 (1987)(hereinafter "Kunkel mutagenesis"), using uracil-containingsingle-stranded DNA template prepared from pHRG2-g3. pHRG2-g3, which isdescribed in Example 1, expressed HRG-β1 177-244 fused to pIII 247. Aseries of oligonucleotides was used to generate a series of constructsthat expressed a series of variants in which consecutive residues weremutated to alanine. Phage stocks were prepared from these constructs asdescribed in Example 1, except that PEG(8000) was used to precipitatethe phage. The affinities of the alanine-substituted variants forErbB-3-Ig and ErbB-4-Ig was determined by phage ELISA as described inExample 1. The results are shown in FIG. 2, which indicates the ratio ofthe EC₅₀ for each variant compared to the EC₅₀ for wild-type HRG-β1177-244, also displayed on phage. In this plot, a ratio of one indicatesthat there was no difference in affinity for variant binding compared towild-type HRG-β1 177-224, and a ratio of, e.g., five indicates that thevariant bound the receptor with an affinity five-fold less than that ofwild-type HRG-β1 177-224.

Expression, Purification, and Assay of Soluble Alanine-SubstitutedVariants

A number of alanine-substituted variants were expressed in soluble formas thioredoxin (Trx) fusion proteins. To prepare suitable expressionvectors, a Trx expression vector was first generated from pET23a(Novagen, Inc., Madison, Wis.). pET23a was digested with NdeI (whichcuts at base 238) and HindIII (which cuts at base 173), and a fragmentencoding Trx was inserted.

This fragment was obtained from pTrxFus (bases 2722-3180; InvitrogenCorp., San Diego, Calif.). The NdeI site, which includes the Trxtranslation start site, was then destroyed by cutting with NdeI andreligating with Klenow. This removed the NdeI site, while retaining theTrx translation start.

Vectors encoding HRG-β1 alanine-substituted variants were initiallygenerated by Kunkel mutagenesis in a pRK5.gDhrgB1 vector (described inGorman et al., DNA Prot. Eng. Tech. 2:2-10 [1990]). The sequencesencoding the variants could be cleaved from these vectors using NdeI andBamHI. To facilitate cloning of such fragments into the Trx expressionvector, Kunkel mutagenesis was used to engineer a KpnI site into thepRK5.gDhrgBl vector encoding wild-type HRG-β1 146-244 immediatelyupstream of the NdeI site (at base 5407). A KpnI-BamHI fragment encodingwild-type HRG-β1 146-224 was then cleaved from pRK5.gDhrgBl and insertedto the Trx expression vector at KpnI and BamHI cloning sites at the 3'end of the sequence encoding Trx. This introduced an NdeI siteimmediately downstream of the KpnI site. In the resultant vector, thewild-type HRG-β1 sequence could be removed by digesting with NdeI andBamHI and replaced with an NdeI-BamHI fragment encoding a variant. Theseries of Trx-variant expression vectors thus obtained expressedTrx-variant fusions that contained an enterokinase protease recognitionsite (DDDDK; SEQ ID NO:37) between the Trx and the variant sequences.

Expression of Trx-variant fusion proteins was driven by the inducible T7promoter from pET23a. Cloning, cell growth, and expression were carriedout as described in the Novagen pET system manual.

Briefly, cloning was done in XL1-Blue™ cells (Stratagene, Inc., LaJolla, Calif.) and expression of soluble protein in BL21DE3 host cells(Novagen, Inc., Madison, Wis.). BL21DE3 cells containing a Trx-variantexpression vector were grown at 37° C. in LE medium until the OD₅₅₀reached 0.3-0.6. Expression of Trx-variant was then induced by additionof 0.4 nM isopropyl-β-D-thiogalactopyranoside (IPTG), and growth wasallowed to continue for 2-4 h at 28° C. Cells were collected bycentrifugation, resuspended in 0.02 M Tris-HCl, 0.025 M EDTA (pH 7.5) toa volume that was 1/20th the cell culture volume.

Cells were lysed by freezing on dry ice, thawing at 37° C., followed byvigorous sonication. The freeze, thaw, and sonication cycle was repeatedthree times. Protein was further solubilized in 6 M GdHCl, 0.1 MTris-HCl (pH 8.8), sulfitolized by the addition of 0.1 M Na₂ SO₃, 0.2 MNa₂ S₄ 0₆, and stirred at room temperature for 1.5 h. Protein wasdialyzed into 0.05 M Tris-HCl (pH 7.5), 0.01 M methionine. Afterdialysis, the insoluble material was removed by centrifugation at 35K×gfor 15 min.

The supernatant was purified by Fast Flow Q Sepharose™ (Pharmacia,Piscataway, N.J.) chromatography using a 15-ml column equilibrated with0.01 M Tris-HCl (pH 7.5). Protein was eluted using a 0-2 M NaCl gradientwith a flow rate of 5 mL/min. The Trx-variant fusions eluted between0.5-0.6 M NaCl and were refolded overnight at room temperature afteraddition of 1 mM cysteine. The resultant preparation was dialyzed into0.05 M Tris-HCl (pH 7.5), 0.01 M methionine. Trx-variant fusions werefound to be essentially homogeneous as determined by amino acid analysisand SDS-PAGE.

The affinities of the Trx-variant fusions for ErbB receptor-Ig fusionswere determined by measuring inhibition of ¹²⁵ I-HRG-β1 177-244 bindingto ErbB-3-Ig and ErbB-4-Ig. Receptor fusions were coated on plates (NuncMaxisorp C™ break-apart strip wells, Inter Med, Denmark) via anti-humanIgG, as described in Example 1 for phage ELISA. Binding assays werecarried out with a constant amount of ¹²⁵ I-HRG-β1 177-244 (100-300 pM)and varying concentrations (100 pM-4 μM) of unlabeled Trx-variantfusion. Following incubation for 1-3 h at room temperature, plates werewashed, and the amount of bound ¹²⁵ I-HRG-β1 177-244 in each well wascounted on a gamma counter (Isodata, ICN Biomedic Systems, Huntsville,Ala.). For the ErbB-3 binding assays, the blocking buffer was TBST(0.025 M Tris-HC1 [pH 7.5], 0.15 M NaCl, 0.02% Tween 20™) containing 1%BSA; the binding buffer was RPMI 1640™ cell culture media (Gibco-BRL,Gaithersburg, Md.) containing 2 mM glutamine, 100 U/mL penicillin, 100μg/mL streptomycin, 10 mM HEPES buffer (pH 7.2), 0.2% BSA; and the washbuffer was TBST. For the ErbB-4 binding assays, PBS containing 1% BSAwas used as the blocking and binding buffers, and the wash buffer wasPBS containing 0.05% Tween 20™.

The results are shown in Tables 4 and 5.

                  TABLE 4                                                         ______________________________________                                        EC.sub.50  Values for Phage and Soluble Alanine-Substituted                     Variant Binding to the ErbB-3 Receptor                                           Variant*    Phage EC.sub.50  (nM)                                                                        Sol EC.sub.50  (nM)                           ______________________________________                                        HRG-β1.sup.#                                                                          13             9                                                   TH-WT@  47.6                                                                  S177 67 129                                                                   H178 >1000 476                                                                L179 ˜1000 394                                                          F189 892 975                                                                  N192 >1000 1642                                                               G193 >1000 26.9                                                               G194 500 1212                                                                 E195 >1000 42                                                                 V199 27                                                                       K200 20 45.4                                                                  P205 22 39.5                                                                  R207 >1000 245                                                                K211 >1000 248                                                                E215 51 156                                                                   F216 500 2550                                                                 T217 >1000 145                                                                G218 >1000 668                                                                R220 >1000 no binding                                                         Y224 865 339                                                                  F229 6 177                                                                  ______________________________________                                         *Variants are identified by the HRGβ1 residues mutated to alanine.       .sup.# HRGB1 is recombinantly produced HRGβ1 177-244.                    .sup.@ THWT is thioredoxin fused at its Cterminus to wildtype HRGβ1      146-244.                                                                 

                  TABLE 5                                                         ______________________________________                                        EC.sub.50  Values for Phage and Soluble Alanine-Substituted                     Variant Binding to the ErbB-4 Receptor                                           Variant*    Phage EC.sub.50  (nM)                                                                        Sol EC.sub.50  (nM)                           ______________________________________                                        HRG-β1.sup.#                                                                          19             14.8                                                TH-WT@  15.4                                                                  S177 83 54.6                                                                  H178 32 138                                                                   L179 56 51.7                                                                  F189 106 565                                                                  N192 188 696                                                                  G193 23 76.3                                                                  G194 65 275                                                                   E195 24 13.2                                                                  V199 47 246                                                                   K200 25 16.9                                                                  P205 8 34.2                                                                   R207 30 24.3                                                                  K211 59 124                                                                   E215 32 104                                                                   F216 >1000 173                                                                T217 14.2 32.7                                                                G218 >1000 608                                                                R220 >1000 no binding                                                         Y224 51 24.5                                                                  F229 >1000 89.9                                                             ______________________________________                                         *Variants are identified by the HRGβ1 residues mutated to alanine.       .sup.# HRGB1 is recombinantly produced HRGβ1 177-244.                    .sup.@ THWT is thioredoxin fused at its Cterminus to wildtype HRGβ1      146-244.                                                                 

EXAMPLE 3 Selection of Heregulin-β1 EGF Domain Variants Using MonovalentPhage Display

This example describes the selection HRG-β1 variants containing residuescorresponding to the minimal EGF-like domain (HRG-β1 177-228). For thesevariants, residue numbers also are expressed, in parentheses, in termsof the position of the residue in the minimal EGF-like domain (i.e.,HRG-β1 EGF 1-52).

Variants of HRG-β1 EGF were prepared and selected for binding toErbB-3-Ig using monovalent phage display, according to the method ofBass et al., Proteins 8:309-14 (1990). As discussed in detail below, anHRG-β1 EGF phagemid vector was prepared, in which HRG-β1 EGF was fusedto a C-terminal fragment of the M13 coat protein pIII. Kunkelmutagenesis was performed to introduce stop codons into this vector atsites selected for randomization. This step ensures that the startingvector is incapable of expressing the wild-type polypeptide. Stretchesof four to six residues per library were randomized in a linear fashion,except for the six cysteines, Phe189 (HRG-β1 EGF Phe13) and the two mostC-terminal residues (see FIG. 3). Phe189 was not altered because thisresidue is conserved as an aromatic residue in EGF and TGF-α and forms astacking interaction with Tyr208 (HRG-β1 EGF Tyr32) Jacobsen et al.,Biochemistry 35:3402-17 (1996). HRG-β1 EGF was thus covered in eightlibraries, designated A-E, G, H and I.

Library E, covering HRG-β1 202-209 (HRG-β1 EGF 26-33), contained athree-residue deletion. The deleted region corresponds to a disorderedturn between the second and third β-sheet of HRG-β1 EGF, and theequivalent amino acids are absent in EGF and TGF-α. An HRG-β1 EGFcontrol variant in which HRG-β1 202-204 (HRG-β1 EGF 26-28) of HRG8 aredeleted (HRG63) bound ErbB-3-Ig with an affinity similar to that ofwild-type (Table 13).

An additional library (F) was created to randomize a surface patchcomposed of side chains from the first and second β-sheets, whichincluded HRG-β1 178, 180, 198, and 200 (HRG-β1 EGF 2, 4, 22, and 24).

The selected sites in the starting vectors were randomized by Kunkelmutagenesis to produce HRG-β1 EGF libraries. Phage displaying mutatedHRG-β1 EGFs were produced from the libraries under conditions such that,statistically, each phage particle displayed no more than one copy ofthe mutated HRG-β1 EGF. See Bass et al., supra. These phage were thenselected for binding to (sorted against) ErbB-3-Ig immobilized on anELISA plate. Bound phage were eluted and used to reinfect host cells,which were used to produce new phage for another round of sorting. Thisprocess was repeated six to seven times for each library. Twelve clonesfrom the phage selected from each library were then sequenced.

Construction of Phage Libraries

Phage libraries were constructed by Kunkel mutagenesis usinguracil-containing single-stranded DNA template prepared from pHRG8-g3.pHRG8-g3, which is described in Example 1, expressed HRG-β1 177-228(HRG-β1 EGF 1-52) fused via a linker to pIII residue 323. For eachlibrary, TAA and TGA stop codons were installed at positions selectedfor randomization to generate custom templates that eliminated wild-typebackground from the pools. Positions were fully randomized by mutationto NNS codons (where N is any of the four bases and S is either G or C).One oligonucleotide was used for each library mutagenesis reactionexcept for Library F, for which two oligonucleotides (one randomizingHRG-β1 178 and 180 [HRG-β1 EGF 2 and 4] and the other randomizingHRG-β-1 198 and 200 [HRG-β1 EGF 22 and 24]), were used simultaneously.Mutagenesis oligonucleotides contained 18-base overhangs on either sideof the randomized residues.

Mutagenesis reaction mixtures were electro-transformed into XL-1 bluecells (Stratagene, Inc., La Jolla, Calif.), according to themanufacturer's protocol. The transformed cells were then infected with10¹¹ pfu M13K07 helper phage (Promega Corp., Madison, Wis.), and phagestocks (about 10¹⁴ phagemid/mL) were prepared as described in Example 1.

Between 1.0×10⁸ and 6.4×10⁸ transformants were obtained for eachlibrary, meaning that the libraries containing five or fewer randomizedcodons had excellent representation of the possible amino acid sequencecombinations (3.36×10⁷ possible DNA sequences; 3.2×10⁶ possible aminoacid sequences). Library B, containing six randomized codons (1.1×10⁹DNA sequences), had 4.8×10⁸ total transformants.

Selection of Phage for ErbB-3-Ig Binding

Monovalent phage were prepared and the selection performed on ErbB-3-Igprebound to microtiter plates via capture with polyclonal antibodies tothe human Fc fragment, as described in Example 1. Approximately 1012phage in 100 μL binding buffer (PBS, 0.1% BSA, 0.05% Tween 20™) wereapplied to an ErbB-3-Ig-coated well and a control well to which noErbB-3-Ig had been added. Following a 2 h incubation at roomtemperature, the plates were washed extensively (12×) and phage elutedby adding 100 μL of a solution of 50 mM HCl and 0.05% Tween™ 20 andshaking for 10 min.

Eluates were neutralized with 10 μL 1 M Tris-HCl (pH 8.0) and 20 μL usedfor titration on log-phase XL-1 blue cells. The remainder was used toinfect 1 mL of log-phase XL-1 blue cells (30 min at 37° C.), which werethen superinfected with 2×10¹⁰ pfu M13KO7 phage and grown in 25 mL 2YTbroth (16 g/L tryptone, 10 g/L yeast extract, 5 g/L NaCl) containing 50μg/mL carbenicillin for 18-24 h. Phage were harvested as described aboveand the cycle repeated. The libraries enriched rapidly, such that byround six of selection the ratio of phage eluted from positive(ErbB-3-Ig-coated) wells to negative (anti-hu Fc precoat only) wells wasbetween 39 and 9200.

After round six (libraries A, B, D-F) or round seven (libraries C, G-I)of selection, twelve clones from each library were randomly picked andsequenced by the dideoxy method. See Sanger et al., PNAS USA 74:5463-67(1977). The amino acids at the randomized positions deduced from the DNAsequences are shown in Tables 6-12 (a "." indicates a residues that isidentical to the wild-type reside). The consensus selected residues ateach position are displayed graphically in FIG. 3. In general, therewere a large number of mutations, in some cases with dramatic changes inthe character of the side chains. At several positions that sorted to aparticular residue, a mixture of DNA codons was found, providingconfidence that the libraries had large diversity and that selection wasat the protein level. In several of the libraries, there was aspontaneous mutation of HRG-β1 Met226Ile (HRG-β1 EGF Met50Ile) due to aone-base change in this codon. This mutation results in a significantaffinity enhancement for ErbB-3-Ig binding. The sequencing results foreach library are summarized below.

Library A--HRG-β1 177-181 (HRG-β1 EGF 1-5)

HRG-β1 177-181 (HRG-β1 EGF 1-5) is present in the first β-strand in thewild-type N-terminal subdomain. The amino acid changes in the variantsselected from this library are shown in Table 6.

                  TABLE 6                                                         ______________________________________                                        Library A Variants                                                                    Position in HRG-β1                                               Variant No.                                                                           177     178    179  180   181                                         ______________________________________                                        Wild-type                                                                             S       H      L    V     K    (SEQ ID NO: 38)                          1 W R . . P (SEQ ID NO: 39)                                                   2 W S . Q P (SEQ ID NO: 40)                                                   3, 5, 10 W E . . P (SEQ ID NO: 41)                                            4 W S . . . (SEQ ID NO: 42)                                                   6 W S . I P (SEQ ID NO: 43)                                                   7 W R . . A (SEQ ID NO: 44)                                                   8 W A . . P (SEQ ID NO: 45)                                                   9 W S . Q . (SEQ ID NO: 46)                                                   11 W E . . A (SEQ ID NO: 47)                                                  12 W S . E P (SEQ ID NO: 48)                                                ______________________________________                                    

Upon randomization, Ser177 (Ser1) sorted exclusively to Trp. His178(His2) sorted to mixed hydrophilic residues, but the wild-type residuewas not among them. Leu179 (Leu3) was conserved in all variantssequenced. Val18O (Val4) sorted to wild-type in eight out of 12variants, and the remaining variants had conservative substitutions atthis position, with the exception of a Val180Glu (Val4Glu) mutation. AtLys181 (Lys5), Pro appeared in eight variants, and the wild-type residuewas found in two variants.

Four variants from library A did not contain amino acid substitutions atpositions randomized in library A. Instead, these variants containedamino acid substitutions at positions randomized in library B, and thusthese variants are listed as variants B1-B4 in Table 7. Similarly, fourvariants from library B did not contain amino acid substitutions atpositions randomized in library B, but rather contained substitutions atpositions randomized in library A. These variants are listed as variantsA5-A8 Table 6.

Library B--HRG-β1 183-188 (HRG-β1 EGF 7-12)

HRG-β1 183-188 (HRG-β1 EGF 7-12) has a helical character in thewild-type protein. The amino acid changes in the variants selected fromthis library are shown in Table 7.

                  TABLE 7                                                         ______________________________________                                        Library B Variants                                                              Variant  Position in HRG-β1                                            No.    183    184    185  186  187  188                                       ______________________________________                                        Wild-type                                                                            A      E      K    E    K    T    (SEQ ID NO: 49)                        1* G V G R D G (SEQ ID NO: 50)                                                2* G G E R E G (SEQ ID NO: 51)                                                3 G . E R E G (SEQ ID NO: 52)                                                 4*, 5* G W D R E G (SEQ ID NO: 53)                                            6* G V Q R E G (SEQ ID NO: 54)                                                7 G . E R A G (SEQ ID NO: 55)                                                 8 G K E R E G (SEQ ID NO: 56)                                                 9* T N S R E G (SEQ ID NO: 57)                                                10* D K S R E G (SEQ ID NO: 58)                                               11* G . D R . Q (SEQ ID NO: 59)                                               12 G R E R E G (SEQ ID NO: 60)                                              ______________________________________                                         *Variant also contained Met226Ile.                                       

Randomization of this region produced the most dramatic changes from thewild-type sequence, although the generally hydrophilic character of thisregion was maintained in the variants sequenced. In particular, thissix-residue stretch sorted to Gly residues at the first and lastpositions, Ala183 and Thr188 (Ala7 and Thr12). There was also a changein registry of positive and negative charges at Lys185 (Lys9), whichsorted to Glu and Asp, among others; Glu186 (Glu10), which sortedexclusively to Arg; and Lys187 (Lys11), which sorted to Glu and Asp.Glu184 (Glu8) sorted to a variety of different types of residues,indicating that this side chain does not play an important role inErbB-3 receptor binding.

Library C--HRG-β1 191-195 (HRG-β1 EGF 15-19)

HRG-β1 191-195 (HRG-β1 EGF 15-19) includes the β-turn between the helixand the second β-strand. The amino acid changes in the variants selectedfrom this library are shown in Table 8.

                  TABLE 8                                                         ______________________________________                                        Library C Variants                                                                     Position in HRG-β1                                              Variant No.                                                                            191    192    193   194  195                                         ______________________________________                                        Wild-type                                                                              V      N      G     G    E    (SEQ ID NO: 61)                          1, 2, 4, 5, 7-12 . . . . . (SEQ ID NO: 62)                                    3 . . . . V (SEQ ID NO: 63)                                                   6 . . . . Q (SEQ ID NO: 64)                                                 ______________________________________                                    

Randomization and selection produced variants in which the wild-typeamino acid sequence in this region was almost completely conserved.Mutations were found in only two variants, both at Glu195 (Glu19). Thisresult is consistent with an important role for the wild-type residuesin this region in ErbB-3 receptor binding.

Library D--HRG-β1 197-201 (HRG-β1 EGF 21-25)

HRG-β1 197-201 (HRG-β1 EGF 21-25) is present in the second β-strand inthe N-terminal subdomain. The amino acid changes in the variantsselected from this library are shown in Table 9.

                  TABLE 9                                                         ______________________________________                                        Library D Variants                                                                     Position in HRG-β1                                              Variant No.                                                                            197    198    199   200  201                                         ______________________________________                                        Wild-type                                                                              F      M      V     K    D    (SEQ ID NO: 65)                          1*, 2*, 8*, 12* Y K . R I (SEQ ID NO: 66)                                     3 . R . . T (SEQ ID NO: 67)                                                   4, 5, 7, 9 Y R . . T (SEQ ID NO: 68)                                          6 Y . I . Y (SEQ ID NO: 69)                                                   10 Y . . . T (SEQ ID NO: 70)                                                  11 M R . R T (SEQ ID NO: 71)                                                ______________________________________                                         *Variant also contained Met226Ile.                                       

Randomization in this region yielded either a gain or loss of charge fortwo of the five residues. Phe197 (Phe21) sorted to Tyr in 10 out of 12variants, maintaining aromaticity at this position. Met198 (Met22)sorted to a positively charged residue in 11 out of 12 variants. Val199(Val23) was conserved in all variants, and Lys200 (Lys24) sorted eitherto wild-type or to Arg, retaining the positive charge at this position.Asp201 (Asp25) sorted to uncharged residues Thr or Ile, retaining theβ-branch character of this position. Variants D1, D2, D8, D12 alsoincluded the spontaneous affinity-enhancing Met226Ile (Met50Ile)mutation.

Library E--HRG-β1 205-209 (HRG-β1 EGF 29-33)

HRG-β1 205-209 (HRG-β1 EGF 29-33) includes residues present in the thirdβ-strand in the N-terminal subdomain. The amino acid changes in thevariants selected from this library are shown in Table 10.

                  TABLE 10                                                        ______________________________________                                        Library E Variants                                                                    Position in HRG-β1                                               Variant No.                                                                           205     206    207  208   209                                         ______________________________________                                        Wild-type                                                                             P       S      R    Y     L    (SEQ ID NO: 72)                          1 T P Y L M (SEQ ID NO: 73)                                                   2, 4 Y G Y L M (SEQ ID NO: 74)                                                3* Y R Y R M (SEQ ID NO: 75)                                                  5, 12 T H Y R G (SEQ ID NO: 76)                                               6 T H Y R M (SEQ ID NO: 77)                                                   7 Y K Y R M (SEQ ID NO: 78)                                                   8, 9 T K Y R G (SEQ ID NO: 79)                                                10 Y K Y R . (SEQ ID NO: 80)                                                  11.sup.#                                                                    ______________________________________                                         *Variant also contained Met226Ile.                                            .sup.# Variant E11 was a contaminant from library F (identical to the         other 12 library F variants).                                            

Upon randomization, Pro205 (Pro29) sorted to Thr or Tyr. Ser206 (Ser30)sorted to mixed residues, predominantly those having basic side chains,although Gly also appears twice (in sequences derived from the samevariant). An inversion of side chains occurred for Arg207 (Arg31) andTyr208 (Tyr32), the first of which sorted exclusively to Tyr and thesecond of which sorted primarily to Arg (seven variants) and Leu (fourvariants). This finding was particularly unexpected, given that Tyr208stacks with Phe189 (Phe13) in the structure and is conserved in the EGFsequence. See Jacobsen et al., Biochemistry 35:3402-17 (1996). At Leu209(Leu33), the relatively conservative Met substitution was found in themajority of variants, but Gly was also found.

Library G--HRG-β1 211-216 (HRG-β1 EGF 35-40)

HRG-β1 211-216 (HRG-β1 EGF 35-40) includes the first β-strand of theC-terminal subdomain of HRG-β1 EGF. The amino acid changes in thevariants selected from this library are shown in Table 11.

                  TABLE 11                                                        ______________________________________                                        Library G Variants                                                              Variant  Position in HRG-β1                                            No.    211    212    213  214  215  216                                       ______________________________________                                        Wild-type                                                                            K      C      P    N    E    F    (SEQ ID NO: 81)                        1, 5, 6, R . S L . . (SEQ ID NO: 82)                                          10, 12                                                                        2 R . S E . . (SEQ ID NO: 83)                                                 3 . . . K . M (SEQ ID NO: 84)                                                 4 R . T V . Y (SEQ ID NO: 85)                                                 7, 8 R . T V . Y (SEQ ID NO: 86)                                              9 . . N S . . (SEQ ID NO: 87)                                                 11 R . K K . . (SEQ ID NO: 88)                                              ______________________________________                                    

Upon randomization, 10 out of 12 variants contained a Lys211Arg(Lys35Arg) mutation, thus retaining a positive charge at this position,which lies between two cysteines. Pro213 (Pro37) sorted to mixedhydrophilic residues, and Asn214 (Asn38) sorted to a mixture ofresidues, with Leu and Val appearing most frequently. Glu215 (Glu39) wasconserved in all variants, and Phe216 (Phe40) was retained in eight outof 12 variants with a conservative Tyr substitution in three of theremaining variants.

Library H--HRG-β1 217-220 (HRG-β1 41-44)

The HRG-β1 217-220 (HRG-β1 EGF 41-44) library proved vulnerable tocontamination by a high-affinity variant from library B (variant B5).This variant was found in 11 out of 12 variants. In the singleunaffected variant, the wild-type amino acid sequence was conservedexcept for an Asp219Glu (Asp43Glu) mutation. This result suggests thatthis region requires the wild-type or similar sequences for optimalbinding.

Library I HRG-β1 222-226 (HRG-β1 EGF 46-50)

HRG-β1 222-226 (HRG-β1 EGF 46-50) includes a short strand of β-sheetthat aligns with the strand of β-sheet at HRG-β1 213-216 (HRG-β1 EGF38-40). The amino acid changes in the variants selected from thislibrary are shown in Table 12.

                  TABLE 12                                                        ______________________________________                                        Library I Variants                                                                     Position in HRG-β1                                              Variant No.                                                                            222    223    224   225  226                                         ______________________________________                                        Wild-type                                                                              Q      N      Y     V    M    (SEQ ID NO: 89)                          1, 4, 8 . W . . I (SEQ ID NO: 90)                                             2, 3, 5-7, 9-12 . H . . I (SEQ ID NO: 91)                                   ______________________________________                                    

Upon randomization of HRG-β1 222-226, only two types of variants werefound in the 12 sequenced, both having wild-type residues conserved atGln222 (Gln46), Tyr224 (Tyr48), and Val225 (Val49), and both having aMet226Ile (Met50Ile) mutation. Asn223 (Asn47) sorted to His (ninevariants) or Trp (three variants). The strong affinity-enhancing effectof Met226Ile is evidenced by its presence in all variants sequenced fromthis library and a high frequency of occurrence in variants from severalother libraries.

Library F--HRG-β1 178, 180, 198, and 200 (HRG-β1 EGF 2, 4, 22 and 24)

When His178 (His2), Val180 (Val4), Met198 (Met22), and Lys200 (Lys24)were simultaneously randomized, only one type of variant was found.His178, Val180, and Lys200 sorted to wild-type residues, and Met198sorted to Lys. These variants additionally contained the spontaneousMet226Ile (Met50Ile) mutation, which gave the variants a significantselective advantage over other sequences. It is striking that thewild-type His was found at position 178 because none of the 12 variantssequenced from library A contained His178.

Conservation of HRG-β1 EGF Residues in Phage Display and AlanineScanning Results

Positions where alanine substitution strongly affected binding affinitytended to sort to the wild-type residue. See, for example, the data forpositions at the junction between the N- and C-terminal subdomains,(i.e., the β-turn at HRG-β1 191-195 [HRG-β1 EGF 15-19] and the loop atHRG-β1 217-220 [HRG-β1 EGF 41-44]) Additionally, positions where alaninesubstitution produced less significant effects tended to undergosubstantial mutation upon phage display, as seen for the helical stretchat HRG-β1 183-188 (HRG-β1 EGF 7-12).

Analysis of the Impact of Selected Mutations on Receptor Affinity andSpecificity

Individual variants from each library were chosen for phage productionand further characterization of the mutated HRG-β1 EGFs ("variants")displayed on the phage. The choice of variants for furthercharacterization was based on selection frequency, with a bias towardssequences not containing the advantageous Met226Ile (Met50Ile)substitution. In addition, phagemid vectors for use in producing phagedisplaying mutated HRG-β1 EGFs containing various combinations of theabove mutations ("combination variants") were prepared by Kunkelmutagenesis, using templates prepared from several of theabove-described variants. The amino acid substitutions in thecombination variants are indicated in FIG. 4. The affinities of thevariants and combination variants for binding to ErbB-3-Ig (relative tothe affinity of wild-type HRG-β1 EGF) were determined by phage ELISA, asdescribed in Example 1. The results are shown in Table 13. Some of thevariants and combination variants were also tested for relativeErbB-4-Ig affinity to assess specificity.

                  TABLE 13                                                        ______________________________________                                        Affinities of Heregulin-β1 EGF Variants and                                Combination Variants for ErbB-3-Ig and ErbB-4-Ig                              as Determined by Phage ELISA                                                              Phage ErbB-3    Phage ErbB-4                                      Construct EC.sub.50  (wt)/EC.sub.50  (mut)* EC.sub.50  (wt)/EC.sub.50                                     (mut)                                           ______________________________________                                        HRG8      1               1                                                     HRG63 1.1 ± 0.8                                                            A1 0.55 ± 0.27                                                             A2 0.87 ± 0.5                                                              A3 0.96 ± 0.43 1.1 ± 0.4                                                A4 <0.3                                                                       B3 4.7 ± 0.77 1.7 ± 0.5                                                 B5 26 ± 20                                                                 B10 9.3                                                                       D1 11 ± 0.45                                                               D4 2.9 ± 1.1 5.0 ± 3.7                                                  D10 2.1                                                                       E2 28 ± 13 7.6                                                             E3 16                                                                         E6 6.6 ± 3.0                                                               E8 17 ± 2.3                                                                G1 1.2 ± 0.36 1.6                                                          G4 0.86 ± 0.19                                                             H5 1.5                                                                        I1 13 ± 11                                                                 12 20 ± 20 10                                                              F1 4.4 ± 3.3                                                               HRG90 6.3 ± 3.1                                                            HRG37 13 ± 15                                                              HRG38 <0.3                                                                    HRG40 <0.3                                                                    HRG41 <0.3                                                                    HRG48 49 ± 15                                                              HRG53 26 ± 16                                                              HRG54 12 ± 9.2                                                             HRG55 13 ± 11                                                              HRG56 31 ± 22                                                              HRG57 24 ± 16                                                              HRG58 58 ± 11 44                                                           HRG59 26 ± 14                                                              HRG60 63 ± 11                                                              HRG61 29 ± 25                                                              HRG62 32 ± 14                                                              HRG71 79 ± 56                                                              HRG73 56 ± 6.6 16                                                        ______________________________________                                         *Based on the wildtype HRGβ1 EGFphage EC.sub.50  (=135 ± 104 nM       for ErbB3-Ig, 163 ± 112 nM for ErbB4-Ig) determined on during the same     assay run. Shown are standard deviations for averages of 2-4 duplicate        runs, or the average only for assays performed in duplicate for one run. 

Variants from library A had EC₅₀ values very similar to wild-type HRG-β1EGF. Variants from libraries B and D had significantly enhanced affinityfor ErbB-3-Ig and ErbB-4-Ig, in the range of three to five times thewild-type affinity for selected variants not containing Met226Ile(Met50Ile). (See variants B3 and D4.) Substantially greater enhancementsin affinity (up to 26-fold wild-type HRG-β1 EGF) were measured forvariants that contained Met226Ile (Met50Ile). (See variants B5, B10, andD1).

Variants from library E showed even greater affinity enhancements. Forexample, variant E2, which differed from the library consensus sequence(FIG. 3) at HRG-β1 206 and 208 (HRG-β1 EGF 30 and 32), had an affinityenhancement of 28-fold. The effects appear to be associated with theamino acid substitutions at HRG-β1 205-209 (HRG-β1 EGF 29-33) ratherthan the adjacent three-residue deletion, since the affinity forErbB-3-Ig of a Δ202-204 (Δ26-28) control variant (HRG63) was similar tothat of wild-type HRG-β1 EGF (Table 13).

The single variant from library F, having Met198Lys (Met22Lys) andMet226Ile (Met50Ile) substitutions, showed an enhancement only slightlyabove that for Met226Ile alone, indicating a small effect attributableto the Met198Lys mutation. Variants from library G showed little, ifany, ErbB-3-Ig affinity enhancement. The single Asp219Glu (Asp43Glu)mutation from the lone library H variant provided a modest increase inaffinity. The two library I variants had significant enhancements inaffinity for ErbB-3-Ig and ErbB-4-Ig. The enhancements were attributableto an approximately six-fold effect from the Met226Ile (Met50Ile)mutation (see variant HRG90), and an additional enhancement of abouttwo- to three-fold from Asn223 (Asn47) to Trp or His mutations.

Phage displaying combination variants containing variant A3 mutationsperformed poorly. The A3+B3 combination had an EC₅₀ for ErbB-3-Igsimilar to that of variant B3, but the other combinations tested did notbind with detectable affinity. This could be due to a disadvantageousinteraction of the Ser177Trp, His178Glu, or Lys181Pro (Ser1Trp, His2Glu,or Lys5Pro) mutations with mutations from the other libraries. Thecombination of the B3 and E2 mutations yielded slightly diminishedaffinities relative to that of variant E2.

The combination of mutations from variants from the other libraries gavecloser to additive behavior and resulted in the enhancement of EC₅₀values by greater than 50-fold. The EC₅₀ values for the best combinationvariants were close to the lower limit of the assay at the receptorcoating concentration used (approximately 4 nM). Using lower levels ofreceptor coat yielded sub-nanomolar EC₅₀ values for these combinationvariants, but did not allow measurement of wild-type affinity, due tominimal binding of wild-type HRG-β1 EGF phage. The best combinationvariants contained the D4 sequence, either the B3 or E2 sequence, andeither the single Met50Ile mutation or the I2 sequence.

In general, variants that exhibited enhancements in affinity forErbB-3-Ig also exhibited similar enhancements in affinity for ErbB-4-Ig.

Expression, Purification, and Assay of Soluble HRG-β1 EGF Variants

A number of variants were expressed in soluble form (i.e., mutatedHRG-β1 EGFs were expressed free of M13 pIII). To facilitate periplasmicexpression of soluble variants, TAG codons were installed followingHRG-β1 226 (HRG-β1 EGF 52) in the corresponding phagemids by Kunkelmutagenesis, and the resulting constructs were transformed into 34B8cells (genotype: tonAΔ phoAΔE15 Δ(argF-lac)169 deoC2 ompTΔ degP41(ΔPstI-kanR)). The constructs included those for expressing variants A3,E2, and F1, and combination variants HRG58 (D4+E2+M50I) andHRG73(B3+D4+E2+I2), as well as the construct that expressed wild-typeHRG-β1 EGF (HRG8).

Expression at levels of about 1 mg/L was achieved as follows. Cellcultures were grown to a density of OD₅₅₀ =1.0 in LB medium containing50 μg/mL carbenicillin and used to inoculate modified AP5 medium at a1/100 dilution. (Modified AP5 medium contained: 1.5 g/L glucose, 11 g/LHycase SF, 0.6 g/L yeast extract, 0.19 g/L anhydrous MgSO₄ or 0.394 g/LMgSO₄ *7H₂ O, 1.07 g/L ammonium chloride, 3.73 g/L KCl, 1.2 g/L NaCl,120 mL/L 1 M triethanolamine [pH 7.4].)

Wild-type HRG-β1 EGF and variants thereof were purified to homogeneityfrom periplasmic shockates in a single reverse-phase HPLC step. Briefly,cells were harvested after 24 h of growth at 30° C. (OD₅₅₀ =1.2) bycentrifugation at 4500 rpm, and the pellets were frozen in ethanol/dryice for 2 h. Following resuspension and thawing in 5 mM MgCl₂, 75 mMCaCl₂, 1 mM phenyl methyl sulfonyl fluoride, and 10 mM Tris-HCl (pH7.6), shocked cells were removed by centrifugation, leaving shockates.Shockates were filtered and chromatographed by semipreparative C18reverse-phase HPLC using a gradient from 0-40% acetonitrile over 80 min,with a flow rate of 3 mL/min. Fractions shown by electrospray massspectrometry to contain HRG-β1 EGF (or variants thereof) werelyophilized and resuspended in 1 mM EDTA, 10 mM Tris-HCl (pH 7.6).Proteins were found to be essentially homogeneous as determined by aminoacid analysis and SDS-PAGE.

The affinities of the soluble variants for ErbB-Ig were determined bymeasuring inhibition of ¹²⁵ I-HRG binding to ErbB-2/3, -3 and -4receptor-Ig fusions, as previously described in Example 1. Although theErbB-2/3-Ig preparation also contains ErbB-2/2 and 3/3 homodimers (as aresult of coexpression of ErbB-2- and -3-Ig's), the displacement of ¹²⁵I-HRG should be predominantly from ErbB-2/3-Ig because of theapproximately 100-fold higher affinity of wild-type HRG-β1 EGF for the2/3-heterodimer versus the 3/3 homodimer.

The results are shown in Table 14.

                  TABLE 14                                                        ______________________________________                                        Binding and Activation Parameters for Soluble                                   Heregulin-β1 EGF Variants                                                          ErbB-3-Ig ErbB-4-Ig                                                                              ErbB-2/3-Ig                                                                            KIRA                                    Construct EC.sub.50 (nM) EC.sub.50 (nM) EC.sub.50 (nM) EC.sub.50            ______________________________________                                                                                (nM)                                  HRG8 (WT)                                                                             2.3 ± 0.2                                                                            1.5 ± 0.1                                                                             0.033 ± 0.003                                                                       0.212                                     A3 4.3 ± 0.2 2.1 ± 0.1  0.067 ± 0.007 0.182                          E2 2.0 ± 0.2 2.4 ± 0.02 0.035 ± 0.007 0.092                          F1 0.60 ± 0.04 0.26 ± 0.011 0.061 ± 0.002 0.151                      HRG58 0.80 ± 0.01 0.30 ± 0.005 0.075 ± 0.006 0.129                   HRG73 2.7 ± 0.1 0.31 ± 0.003  0.20 ± 0.006 0.173                   ______________________________________                                    

The variants showed higher affinity for ErbB-3-Ig than did wild-typeHRG-β1 EGF (up to 4-fold), although the affinity enhancements were lessdramatic than those determined by phage ELISA. Analysis in this formatalso revealed that the E2 mutations confer greatly enhanced affinity,and that the additional D4 and Met50Ile mutations (in construct HRG58)do not contribute additively. As observed in the phage ELISAs, thevariants showed enhancements in affinity for ErbB-4-Ig that were similarto affinity-enhancements for ErbB-3-Ig. Variant affinities forErbB-2/3-Ig were similar to the affinity of wild-type HRG-β1 EGF for the2/3-heterodimer, although the maximally substituted construct (HRG72)bound 6-fold more weakly to ErbB-2/3-Ig than did wild-type HRG-β1 EGF.

The soluble variants were also assayed for their ability to stimulatetyrosine phosphorylation of the ErbB-2 receptor on MCF7 breast cancercarcinoma cells. This was accomplished in a KIRA-ELISA format asdescribed in Sadick et al., Analyt. Biochem. 235:207-214, in which theErbB-2 phosphorylation detected is believed to be due primarily to theformation of ErbB-2/3 heterodimers.

Briefly, 2×10⁵ MCF-7 cells were added to each well of a flat-bottom96-well culture plate and cultured overnight. The following morning theculture medium was replaced with medium containing a variant or HRG8(wild-type) at concentrations ranging from 0 to 10 nM. The cells werestimulated at 37° C. for 30 min, the culture medium was decanted. Tolyse the cells and solubilize the receptors, 100 μl of lysis buffer wasadded to each well. Lysis buffer consisted of 150 mM NaCl, 50 mM HEPES,0.5% Triton-X100, 0.01% thimerosol, 30 kIU/ml aprotinin (ICNBiochemicals, Aurora, Ohio), 1 mM 4-(2-aminoethyl) benzenesulfonylfluoride hydrochloride (AEBSF; ICN Biochemicals), and 2 mM sodiumorthovanadate. The plate was then agitated gently on a plate shaker(Bellco Instruments, Vineland, N.J.) for 60 min at room temperature.

While the cells were being solubilized, an ELISA microtiter plate (NuncMaxisorp™, Inter Med, Denmark) that had been coated overnight at 4° C.with the affinity-purified polyclonal anti-ErbB-2 ECD antibody (1.0μg/ml in 50 mM carbonate buffer (pH 9.6) was decanted, and blocked withblock buffer (PBS, 0.5% BSA [Intergen Co., Purchase, N.Y.], 0.01%thimerosol) for 60 min at room temperature with gentle agitation. After60 min, the anti-ErbB-2 ECD-antibody coated plate was washed six timeswith wash buffer (PBS, 0.05% Tween 20™, 0.01% thimerosol) using anautomated plate washer (ScanWasher 300, Skatron Instruments, Inc.,Sterling, Va.).

The lysate containing solubilized ErbB-2 from each cell-culturemicrotiter well was transferred to each anti-ErbB2 ECD antibody-coatedand blocked ELISA well and incubated for 2 h at room temperature withgentle agitation. The unbound receptor was removed by washing with washbuffer, and 100 μl of biotinylated 4G10 (antiphosphotyrosine antibody)diluted to 0.2 μg/ml in dilution buffer (PBS, 0.5% BSA, 0.05% Tween 20™,5 mM EDTA, 0.01% thimerosol) was added to each well. After incubationfor 2 h at room temperature, the plate was washed, and 100 μlhorseradish peroxidase-conjugated streptavidin (Zymed Laboratories, S.San Francisco, Calif.), diluted 1:50000 in dilution buffer, was added toeach well. The plate was incubated for 30 min at room temperature withgentle agitation. The free avidin conjugate was washed away, and 100 μlfreshly prepared substrate solution (tetramethyl benzidine, TMB,two-component substrate kit, Kirkegard and Perry, Gaithersburg, Md.) wasadded to each well. The reaction was allowed to proceed for 10 min,after which the color development was stopped by the addition of 1.0 MH₃ PO₄.

The results are shown in Table 14. The EC₅₀ is the concentration ofvariant (or HRG8) required to achieve 50% maximal tyrosinephosphorylation. In general, the EC₅₀ 's for stimulation ofphosphorylation by the variants tested did not differ substantially fromthe EC₅₀ for wild-type HRG-β1 EGF. In addition, the values correlatedwell with the IC₅₀ 's for binding to the receptor fusions above.

EXAMPLE 4 Selection of Heregulin-β1 EGF Domain Variants For ErbB-4Receptor Binding Using Monovalent Phage Display

This example describes the selection of HRG-β1 variants for binding tothe ErbB-4 receptor, and in particular, variants having a greaterspecificity for the ErbB-4 receptor, relative to the ErbB-3 receptor,than wild-type HRG-β1. Such variants have, for example, a lowervariant:wild-type EC₅₀ ratio for binding to ErbB-4-Ig than for bindingto ErbB-3-Ig. The variants studied contained residues corresponding toHRG-β1 177-244, which includes the minimal EGF-like domain (HRG-β1177-228). In this example, "HRG-β1 EGF" refers to the region of theEGF-like domain extending from residues 177-244. Residue numbers areexpressed in terms of position in 645-amino acid native human HRG-β1and, in parentheses, in terms of position in HRG-β1 EGF (i.e., HRG-β1EGF 1-68).

The variants were produced by randomization at His178, Leu179, andArg207 (His2, Leu3, and Arg31). These residues were chosen becausealanine scanning (Example 2) indicated that the substitution of alanineat these positions resulted in a significantly greater loss of affinityfor the ErbB-3 receptor, as compared to the ErbB-4 receptor, suggestingthat these residues may be more important for ErbB-3 receptor bindingthan for ErbB-4 receptor binding. Additionally, these residues arepredicted to be proximal to one another on the surface of the HRG-β1molecule, potentially forming a binding site.

Phage Library Screening for ErbB-4-Ig Binding

Variants of HRG-β1 EGF were prepared and selected for binding toErbB-4-Ig using monovalent phage display, according to the method ofBass et al., Proteins 8:309-14 (1990). Briefly, the HRG-β1 EGF phagemidvector was pHRG2-p3 (described in Example 1), in which HRG-β1 177-244was fused to a C-terminal fragment of the M13 coat protein pIII. Kunkelmutagenesis was performed to introduce stop codons into this vector atHis178, Leu179, and Arg207 (His2, Leu3, and Arg31) to ensure that thestarting vector could not express the wild-type polypeptide.

These sites were then randomized by Kunkel mutagenesis to produce anHRG-β1 EGF library. Phage displaying mutated HRG-β1 EGFs monovalentlywere produced from the library. See Bass et al., supra. These phage werethen sorted against ErbB-4-Ig homodimers immobilized on an ELISA plate.Bound phage were eluted and used to reinfect host cells, which were usedto produce new phage. Half of the phage from the first round of sortingwas sorted against immobilized ErbB-4-Ig for three additional rounds ofsorting. The other half of the phage from the first round was sortedagainst immobilized ErbB-4-Ig for three additional rounds in thepresence of soluble ErbB-3-Ig (10 nM). Sorting the presence of solubleErbB-3-Ig (i.e., "counter-selecting" against ErbB-3-Ig) was expected toremove variants with higher affinity for ErbB-3-Ig, allowing enrichmentof those with higher affinity for ErbB-4-Ig in each round of sorting.After sorting, twelve clones from each of the two resultant librarieswere sequenced.

Construction of Initial Phage Library

The initial phage library was constructed by Kunkel mutagenesis usinguracil-containing single-stranded DNA template. TAA and TGA stop codonswere installed at positions selected for randomization to generate acustom template that eliminated wild-type background from the pools.Positions were fully randomized by mutation to NNS codons (where N isany of the four bases and S is either G or C). Two mutagenesisoligonucleotides were used, one to randomize His178 and Leu179 (His2 andLeu3) and one to randomize Arg207 (Arg31). The oligonucleotidescontained 15-base overhangs on either side of the randomized residues.

The final mutagenesis reaction mixture was electro-transformed into XL-1blue cells (Stratagene, Inc., La Jolla, Calif.), according to themanufacturer's protocol. The transformed cells were then infected with10¹¹ pfu M13K07 helper phage (Promega Corp., Madison, Wis.), and phagestocks (about 10¹⁴ phagemid/mL) were prepared as described in Example 1.

At least 10⁸ transformants were obtained, indicating that the libraryhad excellent representation of the possible amino acid sequencecombinations.

Selection of Phage for ErbB-4-Ig Binding

Monovalent phage were prepared and the selection performed on ErbB-4-Igprebound to microtiter plates via capture with polyclonal antibodies tothe human Fc fragment, as described in Example 1. Approximately 10¹²phage in 100 μL binding buffer (PBS, 0.1% BSA, 0.05% Tween 20™) wereapplied to an ErbB-4-Ig-coated well and a control well to which noErbB-4-Ig had been added. After the first round of sorting, half of theresultant phage (hereafter the "counter-selected library") were sortedin the presence of 10 mM ErbB-3-Ig in the binding buffer. Following a 2h incubation at room temperature, the plates were washed extensively(12×) and phage eluted by adding 100 μL of a solution of 50 mM HCl and0.05% Tween™ 20 and shaking for 10 min.

Eluates were neutralized with 10 μL 1 M Tris-HCl (pH 8.0) and 20 μL usedfor titration on log-phase XL-1 blue cells. The remainder was used toinfect 1 mL of log-phase XL-1 blue cells (30 min at 37° C.), which werethen superinfected with 2×10¹⁰ pfu M13KO7 phage and grown in 25 mL 2YTbroth (16 g/L tryptone, 10 g/L yeast extract, 5 g/L NaCl) containing 50μg/mL carbenicillin for 18-24 h. Phage were harvested as described aboveand the cycle repeated.

After round four of selection, twelve clones from each library wererandomly picked and sequenced by the dideoxy method. See Sanger et al.,PNAS USA 74:5463-67 (1977). The amino acids at the randomized positionsdeduced from the DNA sequences are shown in Table 15 (a "." indicates aresidues that is identical to the wild-type residue).

                  TABLE 15                                                        ______________________________________                                        Mutations and Affinities for ErbB-3- and ErbB-4-Igs                             in Phage ELISA                                                                                               ErbB-4   ErbB-3                                Wild-type H L R #Siblings.sup.& EC.sub.50 Mut/Wt.sup.@ EC.sub.50            ______________________________________                                                                                  Mut/WT                              Four Rounds of Sorting for ErbB-4-Ig Binding                                    (No Counter-Selection):                                                       Clones   .     M   .    7      1.5      1.2                                    W . .  2* 1.7 1.2                                                             E . . 2 1.7 9.5                                                               . V . 1 1.6 1.5                                                            Four Rounds of Sorting for ErbB-4-Ig Binding                                    With ErbB-3-Ig Counter-Selection:                                             Clones   .     .   p    7      1.3      6.1                                    L . .  1* 1.7 6.4                                                             L . . 1 1.2 1.4                                                               D V . 1 2.0 2.2                                                               E V . 1 1.7 0.8                                                               T M . 1 1.6 1.4                                                            ______________________________________                                         *HRG-β1 228-331 (HRGβ1 EGF 52-55) were spontaneously deleted an     replaced by a single Met residue.                                             .sup.& Number of variants having this sequence (out of the twelve             sequenced for each library).                                                  .sup.@ EC.sub.50 for the variant divided by the EC.sub.50 for wildtype        HRGβ1 177-244 expressed monovalently on phage                       

One variant predominated in each library, being represented in seven ofthe twelve clones sequenced from each library. The predominant variantfrom the library counter-selected against ErbB-3-Ig sorted back to thewild-type residue at positions His178 and Leu179 (His2 and Leu3) and hadPro at Arg207 (Arg31). The predominant variant from the library that wassorted without counter-selection sorted to wild-type at positions His178and Arg207 (His2 and Arg31) and had Met at Leu179 (Leu3). In variantsfrom each of the libraries, there was a spontaneous substitution of Metfor HRG-β1 228-231 (HRG-β1 EGF 52-55).

Analysis of the Impact of Mutations on Receptor Affinity and Specificity

Receptor binding affinities were measured by phage ELISA for all uniquevariants from each library, as described in Example 1. The results areshown in Table 15. The affinities of the variants for ErbB-3-Ig andErbB-4-Ig were determined to assess specificity.

The affinities of most variants for the ErbB receptor-Ig fusions werereduced between about 1.2-fold and 2.2-fold. Only one variant showed aslight enhancement in affinity, and only for ErbB-3-Ig. However, threevariants had affinities for ErbB-4-Ig that were less than two-fold belowthe affinity of wild-type HRG-β1, whereas their affinities for ErbB-3-Igwere 6- to 9-fold below the affinity of wild-type HRG-β1. All of thesevariants sorted back to the wild-type residue at Leu179 (Leu3). Onevariant sorted to Glu at His178 (His2) and to wild-type at Arg207(Arg31). Another variant sorted to wild-type at His178 (His2) and to Proat Arg 207 (Arg31). This was the predominant variant in thecounter-selected library.

The third variant sorted to Leu at His178 (His2) and to wild-type atArg207 (Arg31) and also had the spontaneous substitution of Met forHRG-β1 228-231 (HRG-β1 EGF 52-55). The affinity of this variant forErbB-3-Ig was reduced 6.4 fold, whereas another variant with the samesequence at His178, Leu179, and Arg207 (His2, Leu3, and Arg31), butlacking the spontaneous substitution, retained approximately wild-typeaffinity for ErbB-3-Ig. Thus, the reduction in affinity for ErbB-3-Igbinding is largely attributable to this spontaneous substitution.Because HRG-β1 228-231 (HRG-β1 EGF 52-55) is unlikely to be proximal tothe randomized region on the surface of the HRG-β1 molecule, the datasuggest that at least two distinct regions of HRG-β1 are important forErbB receptor binding, namely residues His178, Leu179, and Arg207 (His2,Leu3, and Arg31) and HRG-β1 228-231 (HRG-β1 EGF 52-55).

Two of the three variants exhibiting enhanced specificity for ErbB-4-Igbinding accounted for eight of the twelve clones isolated from thecounter-selected library. The other variant accounted for only two ofthe twelve clones isolated from the library that was sorted withoutcounter-selection. Thus, counter-selection against ErbB-3-Ig produced asignificant enrichment in variants exhibiting greater specificity forErbB-4-Ig, relative to ErbB-3-Ig, than that of wild-type HRG-β1.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - <160> NUMBER OF SEQ ID NOS: 116                                      - - <210> SEQ ID NO 1                                                        <211> LENGTH: 71                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                 - - <400> SEQUENCE: 1                                                         - - Ser His Leu Val Lys Cys Ala Glu Lys Glu Ly - #s Thr Phe Cys Val        Asn                                                                              1               5  - #                10  - #                15              - - Gly Gly Glu Cys Phe Met Val Lys Asp Leu Se - #r Asn Pro Ser Arg Tyr                  20      - #            25      - #            30                   - - Leu Cys Lys Cys Pro Asn Glu Phe Thr Gly As - #p Arg Cys Gln Asn Tyr              35          - #        40          - #        45                       - - Val Met Ala Ser Phe Tyr Lys His Leu Gly Il - #e Glu Phe Met Glu Ala          50              - #    55              - #    60                           - - Glu Glu Leu Tyr Gln Lys Arg                                              65                  - #70                                                      - -  - - <210> SEQ ID NO 2                                                   <211> LENGTH: 66                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 2                                                         - - Ser His Leu Val Lys Cys Ala Glu Lys Glu Ly - #s Thr Phe Cys Val Asn       1               5  - #                10  - #                15               - - Gly Gly Glu Cys Phe Met Val Lys Asp Leu Se - #r Asn Pro Ser Arg Tyr                  20      - #            25      - #            30                   - - Leu Cys Lys Cys Gln Pro Gly Phe Thr Gly Al - #a Arg Cys Thr Glu Asn              35          - #        40          - #        45                       - - Val Pro Met Lys Val Gln Asn Gln Glu Lys Al - #a Glu Glu Leu Tyr Gln          50              - #    55              - #    60                           - - Lys Arg                                                                   65                                                                            - -  - - <210> SEQ ID NO 3                                                   <211> LENGTH: 63                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 3                                                         - - Ser His Leu Val Lys Cys Ala Glu Lys Glu Ly - #s Thr Phe Cys Val Asn       1               5  - #                10  - #                15               - - Gly Gly Glu Cys Phe Met Val Lys Asp Leu Se - #r Asn Pro Ser Arg Tyr                  20      - #            25      - #            30                   - - Leu Cys Lys Cys Pro Asn Glu Phe Thr Gly As - #p Arg Cys Gln Asn Tyr              35          - #        40          - #        45                       - - Val Met Ala Ser Phe Tyr Lys Ala Glu Glu Le - #u Tyr Gln Lys Arg              50              - #    55              - #    60                           - -  - - <210> SEQ ID NO 4                                                   <211> LENGTH: 65                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 4                                                         - - Ser His Leu Val Lys Cys Ala Glu Lys Glu Ly - #s Thr Phe Cys Val Asn       1               5  - #                10  - #                15               - - Gly Gly Glu Cys Phe Met Val Lys Asp Leu Se - #r Asn Pro Ser Arg Tyr                  20      - #            25      - #            30                   - - Leu Cys Lys Cys Pro Asn Glu Phe Thr Gly As - #p Arg Cys Gln Asn Tyr              35          - #        40          - #        45                       - - Val Met Ala Ser Phe Tyr Ser Thr Ser Thr Pr - #o Phe Leu Ser Leu Pro          50              - #    55              - #    60                           - - Glu                                                                      65                                                                             - -  - - <210> SEQ ID NO 5                                                   <211> LENGTH: 66                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Rattus rattus                                                  - - <400> SEQUENCE: 5                                                         - - Ser His Leu Ile Lys Cys Ala Glu Lys Glu Ly - #s Thr Phe Cys Val Asn       1               5  - #                10  - #                15               - - Gly Gly Glu Cys Phe Thr Val Lys Asp Leu Se - #r Asn Pro Ser Arg Tyr                  20      - #            25      - #            30                   - - Leu Cys Lys Cys Gln Pro Gly Phe Thr Gly Al - #a Arg Cys Thr Glu Asn              35          - #        40          - #        45                       - - Val Pro Met Lys Val Gln Thr Gln Glu Lys Al - #a Glu Glu Leu Tyr Gln          50              - #    55              - #    60                           - - Lys Arg                                                                  65                                                                             - -  - - <210> SEQ ID NO 6                                                   <211> LENGTH: 71                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Rattus rattus                                                  - - <400> SEQUENCE: 6                                                         - - Ser His Leu Ile Lys Cys Ala Glu Lys Glu Ly - #s Thr Phe Cys Val Asn       1               5  - #                10  - #                15               - - Gly Gly Glu Cys Phe Thr Val Lys Asp Leu Se - #r Asn Pro Ser Arg Tyr                  20      - #            25      - #            30                   - - Leu Cys Lys Cys Pro Asn Glu Phe Thr Gly As - #p Arg Cys Gln Asn Tyr              35          - #        40          - #        45                       - - Val Met Ala Ser Phe Tyr Lys His Leu Gly Il - #e Glu Phe Met Glu Ala          50              - #    55              - #    60                           - - Glu Glu Leu Tyr Gln Lys Arg                                              65                  - #70                                                      - -  - - <210> SEQ ID NO 7                                                   <211> LENGTH: 63                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Rattus rattus                                                  - - <400> SEQUENCE: 7                                                         - - Ser His Leu Ile Lys Cys Ala Glu Lys Glu Ly - #s Thr Phe Cys Val Asn       1               5  - #                10  - #                15               - - Gly Gly Glu Cys Phe Thr Val Lys Asp Leu Se - #r Asn Pro Ser Arg Tyr                  20      - #            25      - #            30                   - - Leu Cys Lys Cys Pro Asn Glu Phe Thr Gly As - #p Arg Cys Gln Asn Tyr              35          - #        40          - #        45                       - - Val Met Ala Ser Phe Tyr Lys Ala Glu Glu Le - #u Tyr Gln Lys Arg              50              - #    55              - #    60                           - -  - - <210> SEQ ID NO 8                                                   <211> LENGTH: 64                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Rattus rattus                                                  - - <400> SEQUENCE: 8                                                         - - Ser His Leu Ile Lys Cys Ala Glu Lys Glu Ly - #s Thr Phe Cys Val Asn       1               5  - #                10  - #                15               - - Gly Gly Glu Cys Phe Thr Val Lys Asp Leu Se - #r Asn Pro Ser Arg Tyr                  20      - #            25      - #            30                   - - Leu Cys Lys Cys Gln Pro Gly Phe Thr Gly Al - #a Arg Cys Thr Glu Asn              35          - #        40          - #        45                       - - Val Pro Met Phe Tyr Ser Thr Ser Thr Pro Ph - #e Leu Ser Leu Pro Glu          50              - #    55              - #    60                           - -  - - <210> SEQ ID NO 9                                                   <211> LENGTH: 81                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Rattus rattus                                                  - - <400> SEQUENCE: 9                                                         - - Ser His Leu Ile Lys Cys Ala Glu Lys Glu Ly - #s Thr Phe Cys Val Asn       1               5  - #                10  - #                15               - - Gly Gly Glu Cys Phe Thr Val Lys Asp Leu Se - #r Asn Pro Ser Arg Tyr                  20      - #            25      - #            30                   - - Leu Cys Lys Cys Gln Pro Gly Phe Thr Gly Al - #a Arg Cys Thr Glu Asn              35          - #        40          - #        45                       - - Val Pro Met Phe Tyr Ser Met Thr Ser Arg Ar - #g Lys Arg Gln Glu Thr          50              - #    55              - #    60                           - - Glu Lys Pro Leu Glu Arg Lys Leu Phe His Se - #r Leu Val Lys Glu Ser      65                  - #70                  - #75                  - #80        - - Lys                                                                       - -  - - <210> SEQ ID NO 10                                                  <211> LENGTH: 65                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 10                                                        - - Ser His Leu Val Lys Cys Ala Glu Lys Glu Ly - #s Thr Phe Cys Val Asn       1               5  - #                10  - #                15               - - Gly Gly Glu Cys Phe Met Val Lys Asp Leu Se - #r Asn Pro Ser Arg Tyr                  20      - #            25      - #            30                   - - Leu Cys Lys Cys Pro Asn Glu Phe Thr Gly As - #p Arg Cys Gln Asn Tyr              35          - #        40          - #        45                       - - Val Met Ala Ser Phe Tyr Ser Thr Ser Thr Pr - #o Phe Leu Ser Leu Pro          50              - #    55              - #    60                           - - Glu                                                                      65                                                                             - -  - - <210> SEQ ID NO 11                                                  <211> LENGTH: 65                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 11                                                        - - Ser His Leu Val Lys Cys Ala Glu Lys Glu Ly - #s Thr Phe Cys Val Asn       1               5  - #                10  - #                15               - - Gly Gly Glu Cys Phe Met Val Lys Asp Leu Se - #r Asn Pro Ser Arg Tyr                  20      - #            25      - #            30                   - - Leu Cys Lys Cys Pro Asn Glu Phe Thr Gly As - #p Arg Cys Gln Asn Tyr              35          - #        40          - #        45                       - - Val Met Ala Ser Phe Tyr Ser Thr Ser Thr Pr - #o Phe Leu Ser Leu Pro          50              - #    55              - #    60                           - - Glu                                                                      65                                                                             - -  - - <210> SEQ ID NO 12                                                  <211> LENGTH: 65                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 12                                                        - - Ser His Leu Val Lys Cys Ala Glu Lys Glu Ly - #s Thr Phe Cys Val Asn       1               5  - #                10  - #                15               - - Gly Gly Glu Cys Phe Met Val Lys Asp Leu Se - #r Asn Pro Ser Arg Tyr                  20      - #            25      - #            30                   - - Leu Cys Lys Cys Pro Asn Glu Phe Thr Gly As - #p Arg Cys Gln Asn Tyr              35          - #        40          - #        45                       - - Val Met Ala Ser Phe Tyr Ser Thr Ser Thr Pr - #o Phe Leu Ser Leu Pro          50              - #    55              - #    60                           - - Glu                                                                      65                                                                             - -  - - <210> SEQ ID NO 13                                                  <211> LENGTH: 71                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Gallus domesticus                                              - - <400> SEQUENCE: 13                                                        - - Ser His Leu Thr Lys Cys Asp Ile Lys Gln Ly - #s Ala Phe Cys Val Asn       1               5  - #                10  - #                15               - - Gly Gly Glu Cys Tyr Met Val Lys Asp Leu Pr - #o Asn Pro Pro Arg Tyr                  20      - #            25      - #            30                   - - Leu Cys Lys Cys Pro Asn Glu Phe Thr Gly As - #p Arg Cys Gln Asn Tyr              35          - #        40          - #        45                       - - Val Met Ala Ser Phe Tyr Lys His Leu Gly Il - #e Glu Phe Met Glu Ala          50              - #    55              - #    60                           - - Glu Glu Leu Tyr Gln Lys Arg                                              65                  - #70                                                      - -  - - <210> SEQ ID NO 14                                                  <211> LENGTH: 49                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Not relevant (recombinant)                                     - - <400> SEQUENCE: 14                                                        - - Ser His Leu Val Lys Cys Ala Glu Lys Glu Ly - #s Thr Phe Cys Val Asn       1               5  - #                10  - #                15               - - Gly Gly Glu Cys Phe Met Val Lys Asp Pro Se - #r Arg Tyr Leu Cys Lys                  20      - #            25      - #            30                   - - Cys Pro Asn Glu Phe Thr Gly Asp Arg Cys Gl - #n Asn Tyr Val Met Ala              35          - #        40          - #        45                       - - Ser                                                                       - -  - - <210> SEQ ID NO 15                                                  <211> LENGTH: 48                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 15                                                        - - Asn Ser Asp Ser Glu Cys Pro Leu Ser His As - #p Gly Tyr Cys Leu His       1               5  - #                10  - #                15               - - Asp Gly Val Cys Met Tyr Ile Glu Ala Leu As - #p Lys Tyr Ala Cys Asn                  20      - #            25      - #            30                   - - Cys Val Val Gly Tyr Ile Gly Glu Arg Cys Gl - #n Tyr Arg Asp Leu Arg              35          - #        40          - #        45                       - -  - - <210> SEQ ID NO 16                                                  <211> LENGTH: 49                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Not relevant (recombinant)                                     - - <400> SEQUENCE: 16                                                        - - Ser His Leu Val Lys Cys Ala Glu Lys Glu Ly - #s Thr Phe Cys Val Asn       1               5  - #                10  - #                15               - - Gly Gly Glu Cys Phe Met Val Lys Asp Pro Se - #r Arg Tyr Leu Cys Lys                  20      - #            25      - #            30                   - - Cys Pro Asn Glu Phe Thr Gly Asp Arg Cys Gl - #n Asn Tyr Val Ile Ala              35          - #        40          - #        45                       - - Ser                                                                       - -  - - <210> SEQ ID NO 17                                                  <211> LENGTH: 52                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Not relevant (recombinant)                                     - - <400> SEQUENCE: 17                                                        - - Trp Glu Leu Val Pro Cys Gly Trp Asp Arg Gl - #u Gly Phe Cys Val Asn       1               5  - #                10  - #                15               - - Gly Gly Glu Cys Phe Met Val Lys Asp Leu Se - #r Asn Pro Ser Arg Tyr                  20      - #            25      - #            30                   - - Leu Cys Lys Cys Pro Asn Glu Phe Thr Gly As - #p Arg Cys Gln Asn Tyr              35          - #        40          - #        45                       - - Val Ile Ala Ser                                                              50                                                                         - -  - - <210> SEQ ID NO 18                                                  <211> LENGTH: 49                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Not relevant (recombinant)                                     - - <400> SEQUENCE: 18                                                        - - Trp Glu Leu Val Pro Cys Ala Glu Lys Glu Ly - #s Thr Phe Cys Val Asn       1               5  - #                10  - #                15               - - Gly Gly Glu Cys Tyr Lys Val Arg Ile Tyr Gl - #y Tyr Leu Met Cys Lys                  20      - #            25      - #            30                   - - Cys Pro Asn Glu Phe Thr Gly Asp Arg Cys Gl - #n Asn Tyr Val Ile Ala              35          - #        40          - #        45                       - - Ser                                                                       - -  - - <210> SEQ ID NO 19                                                  <211> LENGTH: 49                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Not relevant (recombinant)                                     - - <400> SEQUENCE: 19                                                        - - Trp Glu Leu Val Pro Cys Gly Trp Asp Arg Gl - #u Gly Phe Cys Val Asn       1               5  - #                10  - #                15               - - Gly Gly Glu Cys Tyr Lys Val Arg Ile Tyr Gl - #y Tyr Leu Met Cys Lys                  20      - #            25      - #            30                   - - Cys Pro Asn Glu Phe Thr Gly Asp Arg Cys Gl - #n Asn Tyr Val Ile Ala              35          - #        40          - #        45                       - - Ser                                                                       - -  - - <210> SEQ ID NO 20                                                  <211> LENGTH: 49                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Not relevant (recombinant)                                     - - <400> SEQUENCE: 20                                                        - - Trp Glu Leu Val Pro Cys Gly Trp Asp Arg Gl - #u Gly Phe Cys Val Asn       1               5  - #                10  - #                15               - - Gly Gly Glu Cys Tyr Lys Val Arg Ile Tyr Ar - #g Tyr Arg Met Cys Lys                  20      - #            25      - #            30                   - - Cys Pro Asn Glu Phe Thr Gly Asp Arg Cys Gl - #n Asn Tyr Val Ile Ala              35          - #        40          - #        45                       - - Ser                                                                       - -  - - <210> SEQ ID NO 21                                                  <211> LENGTH: 49                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Not relevant (recombinant)                                     - - <400> SEQUENCE: 21                                                        - - Ser His Leu Val Lys Cys Ala Glu Lys Glu Ly - #s Thr Phe Cys Val Asn       1               5  - #                10  - #                15               - - Gly Gly Glu Cys Phe Met Val Lys Asp Tyr Gl - #y Tyr Leu Met Cys Lys                  20      - #            25      - #            30                   - - Cys Pro Asn Glu Phe Thr Gly Asp Arg Cys Gl - #n Asn Tyr Val Ile Ala              35          - #        40          - #        45                       - - Ser                                                                       - -  - - <210> SEQ ID NO 22                                                  <211> LENGTH: 52                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Not relevant (recombinant)                                     - - <400> SEQUENCE: 22                                                        - - Ser His Leu Val Lys Cys Gly Glu Glu Arg Gl - #u Gly Phe Cys Val Asn       1               5  - #                10  - #                15               - - Gly Gly Glu Cys Tyr Arg Val Lys Thr Leu Se - #r Asn Pro Ser Arg Tyr                  20      - #            25      - #            30                   - - Leu Cys Lys Cys Pro Asn Glu Phe Thr Gly As - #p Arg Cys Gln Asn Tyr              35          - #        40          - #        45                       - - Val Met Ala Ser                                                              50                                                                         - -  - - <210> SEQ ID NO 23                                                  <211> LENGTH: 49                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Not relevant (recombinant)                                     - - <400> SEQUENCE: 23                                                        - - Ser His Leu Val Lys Cys Gly Glu Glu Arg Gl - #u Gly Phe Cys Val Asn       1               5  - #                10  - #                15               - - Gly Gly Glu Cys Phe Met Val Lys Asp Tyr Gl - #y Tyr Leu Met Cys Lys                  20      - #            25      - #            30                   - - Cys Pro Asn Glu Phe Thr Gly Asp Arg Cys Gl - #n Asn Tyr Val Met Ala              35          - #        40          - #        45                       - - Ser                                                                       - -  - - <210> SEQ ID NO 24                                                  <211> LENGTH: 49                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Not relevant (recombinant)                                     - - <400> SEQUENCE: 24                                                        - - Ser His Leu Val Lys Cys Gly Glu Glu Arg Gl - #u Gly Phe Cys Val Asn       1               5  - #                10  - #                15               - - Gly Gly Glu Cys Tyr Arg Val Lys Thr Tyr Gl - #y Tyr Leu Met Cys Lys                  20      - #            25      - #            30                   - - Cys Pro Asn Glu Phe Thr Gly Asp Arg Cys Gl - #n Asn Tyr Val Met Ala              35          - #        40          - #        45                       - - Ser                                                                       - -  - - <210> SEQ ID NO 25                                                  <211> LENGTH: 52                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Not relevant (recombinant)                                     - - <400> SEQUENCE: 25                                                        - - Ser His Leu Val Lys Cys Gly Glu Glu Arg Gl - #u Gly Phe Cys Val Asn       1               5  - #                10  - #                15               - - Gly Gly Glu Cys Phe Met Val Lys Asp Leu Se - #r Asn Pro Ser Arg Tyr                  20      - #            25      - #            30                   - - Leu Cys Lys Cys Pro Asn Glu Phe Thr Gly As - #p Arg Cys Gln Asn Tyr              35          - #        40          - #        45                       - - Val Ile Ala Ser                                                              50                                                                         - -  - - <210> SEQ ID NO 26                                                  <211> LENGTH: 49                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Not relevant (recombinant)                                     - - <400> SEQUENCE: 26                                                        - - Ser His Leu Val Lys Cys Ala Glu Lys Glu Ly - #s Thr Phe Cys Val Asn       1               5  - #                10  - #                15               - - Gly Gly Glu Cys Tyr Arg Val Lys Thr Tyr Gl - #y Tyr Leu Met Cys Lys                  20      - #            25      - #            30                   - - Cys Pro Asn Glu Phe Thr Gly Asp Arg Cys Gl - #n Asn Tyr Val Met Ala              35          - #        40          - #        45                       - - Ser                                                                       - -  - - <210> SEQ ID NO 27                                                  <211> LENGTH: 49                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Not relevant (recombinant)                                     - - <400> SEQUENCE: 27                                                        - - Ser His Leu Val Lys Cys Ala Glu Lys Glu Ly - #s Thr Phe Cys Val Asn       1               5  - #                10  - #                15               - - Gly Gly Glu Cys Tyr Arg Val Lys Thr Tyr Gl - #y Tyr Leu Met Cys Lys                  20      - #            25      - #            30                   - - Cys Pro Asn Glu Phe Thr Gly Asp Arg Cys Gl - #n Asn Tyr Val Ile Ala              35          - #        40          - #        45                       - - Ser                                                                       - -  - - <210> SEQ ID NO 28                                                  <211> LENGTH: 52                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Not relevant (recombinant)                                     - - <400> SEQUENCE: 28                                                        - - Ser His Leu Val Lys Cys Ala Glu Lys Glu Ly - #s Thr Phe Cys Val Asn       1               5  - #                10  - #                15               - - Gly Gly Glu Cys Tyr Arg Val Lys Thr Leu Se - #r Asn Pro Ser Arg Tyr                  20      - #            25      - #            30                   - - Leu Cys Lys Cys Pro Asn Glu Phe Thr Gly As - #p Arg Cys Gln Asn Tyr              35          - #        40          - #        45                       - - Val Ile Ala Ser                                                              50                                                                         - -  - - <210> SEQ ID NO 29                                                  <211> LENGTH: 52                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Not relevant (recombinant)                                     - - <400> SEQUENCE: 29                                                        - - Ser His Leu Val Lys Cys Gly Glu Glu Arg Gl - #u Gly Phe Cys Val Asn       1               5  - #                10  - #                15               - - Gly Gly Glu Cys Tyr Arg Val Lys Thr Leu Se - #r Asn Pro Ser Arg Tyr                  20      - #            25      - #            30                   - - Leu Cys Lys Cys Pro Asn Glu Phe Thr Gly As - #p Arg Cys Gln Asn Tyr              35          - #        40          - #        45                       - - Val Ile Ala Ser                                                              50                                                                         - -  - - <210> SEQ ID NO 30                                                  <211> LENGTH: 49                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Not relevant (recombinant)                                     - - <400> SEQUENCE: 30                                                        - - Ser His Leu Val Lys Cys Gly Glu Glu Arg Gl - #u Gly Phe Cys Val Asn       1               5  - #                10  - #                15               - - Gly Gly Glu Cys Phe Met Val Lys Asp Tyr Gl - #y Tyr Leu Met Cys Lys                  20      - #            25      - #            30                   - - Cys Pro Asn Glu Phe Thr Gly Asp Arg Cys Gl - #n Asn Tyr Val Ile Ala              35          - #        40          - #        45                       - - Ser                                                                       - -  - - <210> SEQ ID NO 31                                                  <211> LENGTH: 49                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Not relevant (recombinant)                                     - - <400> SEQUENCE: 31                                                        - - Ser His Leu Val Lys Cys Gly Glu Glu Arg Gl - #u Gly Phe Cys Val Asn       1               5  - #                10  - #                15               - - Gly Gly Glu Cys Tyr Arg Val Lys Thr Tyr Gl - #y Tyr Leu Met Cys Lys                  20      - #            25      - #            30                   - - Cys Pro Asn Glu Phe Thr Gly Asp Arg Cys Gl - #n Asn Tyr Val Ile Ala              35          - #        40          - #        45                       - - Ser                                                                       - -  - - <210> SEQ ID NO 32                                                  <211> LENGTH: 49                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Not relevant (recombinant)                                     - - <400> SEQUENCE: 32                                                        - - Ser His Leu Val Lys Cys Ala Glu Lys Glu Ly - #s Thr Phe Cys Val Asn       1               5  - #                10  - #                15               - - Gly Gly Glu Cys Tyr Arg Val Lys Thr Tyr Gl - #y Tyr Leu Met Cys Lys                  20      - #            25      - #            30                   - - Cys Pro Asn Glu Phe Thr Gly Asp Arg Cys Gl - #n His Tyr Val Ile Ala              35          - #        40          - #        45                       - - Ser                                                                       - -  - - <210> SEQ ID NO 33                                                  <211> LENGTH: 49                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Not relevant (recombinant)                                     - - <400> SEQUENCE: 33                                                        - - Ser His Leu Val Lys Cys Gly Glu Glu Arg Gl - #u Gly Phe Cys Val Asn       1               5  - #                10  - #                15               - - Gly Gly Glu Cys Tyr Arg Val Lys Thr Tyr Gl - #y Tyr Leu Met Cys Lys                  20      - #            25      - #            30                   - - Cys Pro Asn Glu Phe Thr Gly Asp Arg Cys Gl - #n His Tyr Val Ile Ala              35          - #        40          - #        45                       - - Ser                                                                       - -  - - <210> SEQ ID NO 34                                                  <211> LENGTH: 4                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Not relevant (recombinant)                                     - - <400> SEQUENCE: 34                                                        - - Gly Gly Gly Ser                                                           1                                                                             - -  - - <210> SEQ ID NO 35                                                  <211> LENGTH: 7                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Not relevant (recombinant)                                     - - <400> SEQUENCE: 35                                                        - - Gly Gly Gly Ser Gly Gly Gly                                               1               5                                                             - -  - - <210> SEQ ID NO 36                                                  <211> LENGTH: 5                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Not relevant (recombinant)                                     - - <400> SEQUENCE: 36                                                        - - Thr Arg Asp Lys Thr                                                       1               5                                                             - -  - - <210> SEQ ID NO 37                                                  <211> LENGTH: 5                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Not relevant (recombinant)                                     - - <400> SEQUENCE: 37                                                        - - Asp Asp Asp Asp Lys                                                       1               5                                                             - -  - - <210> SEQ ID NO 38                                                  <211> LENGTH: 5                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 38                                                        - - Ser His Leu Val Lys                                                       1               5                                                             - -  - - <210> SEQ ID NO 39                                                  <211> LENGTH: 5                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Not relevant (recombinant)                                     - - <400> SEQUENCE: 39                                                        - - Trp Arg Leu Val Pro                                                       1               5                                                             - -  - - <210> SEQ ID NO 40                                                  <211> LENGTH: 5                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Not relevant (recombinant)                                     - - <400> SEQUENCE: 40                                                        - - Trp Ser Leu Gln Pro                                                       1               5                                                             - -  - - <210> SEQ ID NO 41                                                  <211> LENGTH: 5                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Not relevant (recombinant)                                     - - <400> SEQUENCE: 41                                                        - - Trp Glu Leu Val Pro                                                       1               5                                                             - -  - - <210> SEQ ID NO 42                                                  <211> LENGTH: 5                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Not relevant (recombinant)                                     - - <400> SEQUENCE: 42                                                        - - Trp Ser Leu Val Lys                                                       1               5                                                             - -  - - <210> SEQ ID NO 43                                                  <211> LENGTH: 5                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Not relevant (recombinant)                                     - - <400> SEQUENCE: 43                                                        - - Trp Ser Leu Ile Pro                                                       1               5                                                             - -  - - <210> SEQ ID NO 44                                                  <211> LENGTH: 5                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Not relevant (recombinant)                                     - - <400> SEQUENCE: 44                                                        - - Trp Arg Leu Val Ala                                                       1               5                                                             - -  - - <210> SEQ ID NO 45                                                  <211> LENGTH: 5                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Not relevant (recombinant)                                     - - <400> SEQUENCE: 45                                                        - - Trp Ala Leu Val Pro                                                       1               5                                                             - -  - - <210> SEQ ID NO 46                                                  <211> LENGTH: 5                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Not relevant (recombinant)                                     - - <400> SEQUENCE: 46                                                        - - Trp Ser Leu Gln Lys                                                       1               5                                                             - -  - - <210> SEQ ID NO 47                                                  <211> LENGTH: 5                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Not relevant (recombinant)                                     - - <400> SEQUENCE: 47                                                        - - Trp Glu Leu Val Ala                                                       1               5                                                             - -  - - <210> SEQ ID NO 48                                                  <211> LENGTH: 5                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Not relevant (recombinant)                                     - - <400> SEQUENCE: 48                                                        - - Trp Ser Leu Glu Pro                                                       1               5                                                             - -  - - <210> SEQ ID NO 49                                                  <211> LENGTH: 6                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 49                                                        - - Ala Glu Lys Glu Lys Thr                                                   1               5                                                             - -  - - <210> SEQ ID NO 50                                                  <211> LENGTH: 6                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Not relevant (recombinant)                                     - - <400> SEQUENCE: 50                                                        - - Gly Val Gly Arg Asp Gly                                                   1               5                                                             - -  - - <210> SEQ ID NO 51                                                  <211> LENGTH: 6                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Not relevant (recombinant)                                     - - <400> SEQUENCE: 51                                                        - - Gly Gly Glu Arg Glu Gly                                                   1               5                                                             - -  - - <210> SEQ ID NO 52                                                  <211> LENGTH: 6                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Not relevant (recombinant)                                     - - <400> SEQUENCE: 52                                                        - - Gly Glu Glu Arg Glu Gly                                                   1               5                                                             - -  - - <210> SEQ ID NO 53                                                  <211> LENGTH: 6                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Not relevant (recombinant)                                     - - <400> SEQUENCE: 53                                                        - - Gly Trp Asp Arg Glu Gly                                                   1               5                                                             - -  - - <210> SEQ ID NO 54                                                  <211> LENGTH: 6                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Not relevant (recombinant)                                     - - <400> SEQUENCE: 54                                                        - - Gly Val Gln Arg Glu Gly                                                   1               5                                                             - -  - - <210> SEQ ID NO 55                                                  <211> LENGTH: 6                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Not relevant (recombinant)                                     - - <400> SEQUENCE: 55                                                        - - Gly Glu Glu Arg Ala Gly                                                   1               5                                                             - -  - - <210> SEQ ID NO 56                                                  <211> LENGTH: 6                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Not relevant (recombinant)                                     - - <400> SEQUENCE: 56                                                        - - Gly Lys Glu Arg Glu Gly                                                   1               5                                                             - -  - - <210> SEQ ID NO 57                                                  <211> LENGTH: 6                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Not relevant (recombinant)                                     - - <400> SEQUENCE: 57                                                        - - Thr Asn Ser Arg Glu Gly                                                   1               5                                                             - -  - - <210> SEQ ID NO 58                                                  <211> LENGTH: 6                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Not relevant (recombinant)                                     - - <400> SEQUENCE: 58                                                        - - Asp Lys Ser Arg Glu Gly                                                   1               5                                                             - -  - - <210> SEQ ID NO 59                                                  <211> LENGTH: 6                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Not relevant (recombinant)                                     - - <400> SEQUENCE: 59                                                        - - Gly Glu Asp Arg Lys Gln                                                   1               5                                                             - -  - - <210> SEQ ID NO 60                                                  <211> LENGTH: 6                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Not relevant (recombinant)                                     - - <400> SEQUENCE: 60                                                        - - Gly Arg Glu Arg Glu Gly                                                   1               5                                                             - -  - - <210> SEQ ID NO 61                                                  <211> LENGTH: 5                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 61                                                        - - Val Asn Gly Gly Glu                                                       1               5                                                             - -  - - <210> SEQ ID NO 62                                                  <211> LENGTH: 5                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Not relevant (recombinant)                                     - - <400> SEQUENCE: 62                                                        - - Val Asn Gly Gly Glu                                                       1               5                                                             - -  - - <210> SEQ ID NO 63                                                  <211> LENGTH: 5                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Not relevant (recombinant)                                     - - <400> SEQUENCE: 63                                                        - - Val Asn Gly Gly Val                                                       1               5                                                             - -  - - <210> SEQ ID NO 64                                                  <211> LENGTH: 5                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Not relevant (recombinant)                                     - - <400> SEQUENCE: 64                                                        - - Val Asn Gly Gly Gln                                                       1               5                                                             - -  - - <210> SEQ ID NO 65                                                  <211> LENGTH: 5                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 65                                                        - - Phe Met Val Lys Asp                                                       1               5                                                             - -  - - <210> SEQ ID NO 66                                                  <211> LENGTH: 5                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Not relevant (recombinant)                                     - - <400> SEQUENCE: 66                                                        - - Tyr Lys Val Arg Ile                                                       1               5                                                             - -  - - <210> SEQ ID NO 67                                                  <211> LENGTH: 5                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Not relevant (recombinant)                                     - - <400> SEQUENCE: 67                                                        - - Phe Arg Val Lys Thr                                                       1               5                                                             - -  - - <210> SEQ ID NO 68                                                  <211> LENGTH: 5                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Not relevant (recombinant)                                     - - <400> SEQUENCE: 68                                                        - - Tyr Arg Val Lys Thr                                                       1               5                                                             - -  - - <210> SEQ ID NO 69                                                  <211> LENGTH: 5                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Not relevant (recombinant)                                     - - <400> SEQUENCE: 69                                                        - - Tyr Met Ile Lys Tyr                                                       1               5                                                             - -  - - <210> SEQ ID NO 70                                                  <211> LENGTH: 5                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Not relevant (recombinant)                                     - - <400> SEQUENCE: 70                                                        - - Tyr Met Val Lys Thr                                                       1               5                                                             - -  - - <210> SEQ ID NO 71                                                  <211> LENGTH: 5                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Not relevant (recombinant)                                     - - <400> SEQUENCE: 71                                                        - - Met Arg Val Arg Thr                                                       1               5                                                             - -  - - <210> SEQ ID NO 72                                                  <211> LENGTH: 5                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Not relevant (recombinant)                                     - - <400> SEQUENCE: 72                                                        - - Pro Ser Arg Tyr Leu                                                       1               5                                                             - -  - - <210> SEQ ID NO 73                                                  <211> LENGTH: 5                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Not relevant (recombinant)                                     - - <400> SEQUENCE: 73                                                        - - Thr Pro Tyr Leu Met                                                       1               5                                                             - -  - - <210> SEQ ID NO 74                                                  <211> LENGTH: 5                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Not relevant (recombinant)                                     - - <400> SEQUENCE: 74                                                        - - Tyr Gly Tyr Leu Met                                                       1               5                                                             - -  - - <210> SEQ ID NO 75                                                  <211> LENGTH: 5                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Not relevant (recombinant)                                     - - <400> SEQUENCE: 75                                                        - - Tyr Arg Tyr Arg Met                                                       1               5                                                             - -  - - <210> SEQ ID NO 76                                                  <211> LENGTH: 5                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Not relevant (recombinant)                                     - - <400> SEQUENCE: 76                                                        - - Thr His Tyr Arg Gly                                                       1               5                                                             - -  - - <210> SEQ ID NO 77                                                  <211> LENGTH: 5                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Not relevant (recombinant)                                     - - <400> SEQUENCE: 77                                                        - - Thr His Tyr Arg Met                                                       1               5                                                             - -  - - <210> SEQ ID NO 78                                                  <211> LENGTH: 5                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Not relevant (recombinant)                                     - - <400> SEQUENCE: 78                                                        - - Tyr Lys Tyr Arg Met                                                       1               5                                                             - -  - - <210> SEQ ID NO 79                                                  <211> LENGTH: 5                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Not relevant (recombinant)                                     - - <400> SEQUENCE: 79                                                        - - Thr Lys Tyr Arg Gly                                                       1               5                                                             - -  - - <210> SEQ ID NO 80                                                  <211> LENGTH: 5                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Not relevant (recombinant)                                     - - <400> SEQUENCE: 80                                                        - - Tyr Lys Tyr Arg Leu                                                       1               5                                                             - -  - - <210> SEQ ID NO 81                                                  <211> LENGTH: 6                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 81                                                        - - Lys Cys Pro Asn Glu Phe                                                   1               5                                                             - -  - - <210> SEQ ID NO 82                                                  <211> LENGTH: 6                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Not relevant (recombinant)                                     - - <400> SEQUENCE: 82                                                        - - Arg Cys Ser Leu Glu Phe                                                   1               5                                                             - -  - - <210> SEQ ID NO 83                                                  <211> LENGTH: 6                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Not relevant (recombinant)                                     - - <400> SEQUENCE: 83                                                        - - Arg Cys Ser Glu Glu Phe                                                   1               5                                                             - -  - - <210> SEQ ID NO 84                                                  <211> LENGTH: 6                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Not relevant (recombinant)                                     - - <400> SEQUENCE: 84                                                        - - Lys Cys Pro Lys Glu Met                                                   1               5                                                             - -  - - <210> SEQ ID NO 85                                                  <211> LENGTH: 6                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Not relevant (recombinant)                                     - - <400> SEQUENCE: 85                                                        - - Arg Cys Thr Val Glu Tyr                                                   1               5                                                             - -  - - <210> SEQ ID NO 86                                                  <211> LENGTH: 6                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Not relevant (recombinant)                                     - - <400> SEQUENCE: 86                                                        - - Arg Cys Thr Val Glu Tyr                                                   1               5                                                             - -  - - <210> SEQ ID NO 87                                                  <211> LENGTH: 6                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Not relevant (recombinant)                                     - - <400> SEQUENCE: 87                                                        - - Lys Cys Asn Ser Glu Phe                                                   1               5                                                             - -  - - <210> SEQ ID NO 88                                                  <211> LENGTH: 6                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Not relevant (recombinant)                                     - - <400> SEQUENCE: 88                                                        - - Arg Cys Lys Lys Glu Phe                                                   1               5                                                             - -  - - <210> SEQ ID NO 89                                                  <211> LENGTH: 5                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 89                                                        - - Gln Asn Tyr Val Met                                                       1               5                                                             - -  - - <210> SEQ ID NO 90                                                  <211> LENGTH: 5                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Not relevant (recombinant)                                     - - <400> SEQUENCE: 90                                                        - - Gln Trp Tyr Val Ile                                                       1               5                                                             - -  - - <210> SEQ ID NO 91                                                  <211> LENGTH: 5                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Not relevant (recombinant)                                     - - <400> SEQUENCE: 91                                                        - - Gln His Tyr Val Ile                                                       1               5                                                             - -  - - <210> SEQ ID NO 92                                                  <211> LENGTH: 52                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 92                                                        - - Ser His Leu Val Lys Cys Ala Glu Lys Glu Ly - #s Thr Phe Cys Val Asn       1               5  - #                10  - #                15               - - Gly Gly Glu Cys Phe Met Val Lys Asp Leu Se - #r Asn Pro Ser Arg Tyr                  20      - #            25      - #            30                   - - Leu Cys Lys Cys Pro Asn Glu Phe Thr Gly As - #p Arg Cys Gln Asn Tyr              35          - #        40          - #        45                       - - Val Met Ala Ser                                                              50                                                                         - -  - - <210> SEQ ID NO 93                                                  <211> LENGTH: 645                                                             <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 93                                                        - - Met Ser Glu Arg Lys Glu Gly Arg Gly Lys Gl - #y Lys Gly Lys Lys Lys       1               5  - #                10  - #                15               - - Glu Arg Gly Ser Gly Lys Lys Pro Glu Ser Al - #a Ala Gly Ser Gln Ser                  20      - #            25      - #            30                   - - Pro Ala Leu Pro Pro Gln Leu Lys Glu Met Ly - #s Ser Gln Glu Ser Ala              35          - #        40          - #        45                       - - Ala Gly Ser Lys Leu Val Leu Arg Cys Glu Th - #r Ser Ser Glu Tyr Ser          50              - #    55              - #    60                           - - Ser Leu Arg Phe Lys Trp Phe Lys Asn Gly As - #n Glu Leu Asn Arg Lys      65                  - #70                  - #75                  - #80        - - Asn Lys Pro Gln Asn Ile Lys Ile Gln Lys Ly - #s Pro Gly Lys Ser Glu                      85  - #                90  - #                95               - - Leu Arg Ile Asn Lys Ala Ser Leu Ala Asp Se - #r Gly Glu Tyr Met Cys                  100      - #           105      - #           110                  - - Lys Val Ile Ser Lys Leu Gly Asn Asp Ser Al - #a Ser Ala Asn Ile Thr              115          - #       120          - #       125                      - - Ile Val Glu Ser Asn Glu Ile Ile Thr Gly Me - #t Pro Ala Ser Thr Glu          130              - #   135              - #   140                          - - Gly Ala Tyr Val Ser Ser Glu Ser Pro Ile Ar - #g Ile Ser Val Ser Thr      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Glu Gly Ala Asn Thr Ser Ser Ser Thr Ser Th - #r Ser Thr Thr Gly        Thr                                                                                             165  - #               170  - #               175             - - Ser His Leu Val Lys Cys Ala Glu Lys Glu Ly - #s Thr Phe Cys Val Asn                  180      - #           185      - #           190                  - - Gly Gly Glu Cys Phe Met Val Lys Asp Leu Se - #r Asn Pro Ser Arg Tyr              195          - #       200          - #       205                      - - Leu Cys Lys Cys Pro Asn Glu Phe Thr Gly As - #p Arg Cys Gln Asn Tyr          210              - #   215              - #   220                          - - Val Met Ala Ser Phe Tyr Lys His Leu Gly Il - #e Glu Phe Met Glu Ala      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Glu Glu Leu Tyr Gln Lys Arg Val Leu Thr Il - #e Thr Gly Ile Cys        Ile                                                                                             245  - #               250  - #               255             - - Ala Leu Leu Val Val Gly Ile Met Cys Val Va - #l Ala Tyr Cys Lys Thr                  260      - #           265      - #           270                  - - Lys Lys Gln Arg Lys Lys Leu His Asp Arg Le - #u Arg Gln Ser Leu Arg              275          - #       280          - #       285                      - - Ser Glu Arg Asn Asn Met Met Asn Ile Ala As - #n Gly Pro His His Pro          290              - #   295              - #   300                          - - Asn Pro Pro Pro Glu Asn Val Gln Leu Val As - #n Gln Tyr Val Ser Lys      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Asn Val Ile Ser Ser Glu His Ile Val Glu Ar - #g Glu Ala Glu Thr        Ser                                                                                             325  - #               330  - #               335             - - Phe Ser Thr Ser His Tyr Thr Ser Thr Ala Hi - #s His Ser Thr Thr Val                  340      - #           345      - #           350                  - - Thr Gln Thr Pro Ser His Ser Trp Ser Asn Gl - #y His Thr Glu Ser Ile              355          - #       360          - #       365                      - - Leu Ser Glu Ser His Ser Val Ile Val Met Se - #r Ser Val Glu Asn Ser          370              - #   375              - #   380                          - - Arg His Ser Ser Pro Thr Gly Gly Pro Arg Gl - #y Arg Leu Asn Gly Thr      385                 3 - #90                 3 - #95                 4 -      #00                                                                              - - Gly Gly Pro Arg Glu Cys Asn Ser Phe Leu Ar - #g His Ala Arg Glu        Thr                                                                                             405  - #               410  - #               415             - - Pro Asp Ser Tyr Arg Asp Ser Pro His Ser Gl - #u Arg Tyr Val Ser Ala                  420      - #           425      - #           430                  - - Met Thr Thr Pro Ala Arg Met Ser Pro Val As - #p Phe His Thr Pro Ser              435          - #       440          - #       445                      - - Ser Pro Lys Ser Pro Pro Ser Glu Met Ser Pr - #o Pro Val Ser Ser Met          450              - #   455              - #   460                          - - Thr Val Ser Met Pro Ser Met Ala Val Ser Pr - #o Phe Met Glu Glu Glu      465                 4 - #70                 4 - #75                 4 -      #80                                                                              - - Arg Pro Leu Leu Leu Val Thr Pro Pro Arg Le - #u Arg Glu Lys Lys        Phe                                                                                             485  - #               490  - #               495             - - Asp His His Pro Gln Gln Phe Ser Ser Phe Hi - #s His Asn Pro Ala His                  500      - #           505      - #           510                  - - Asp Ser Asn Ser Leu Pro Ala Ser Pro Leu Ar - #g Ile Val Glu Asp Glu              515          - #       520          - #       525                      - - Glu Tyr Glu Thr Thr Gln Glu Tyr Glu Pro Al - #a Gln Glu Pro Val Lys          530              - #   535              - #   540                          - - Lys Leu Ala Asn Ser Arg Arg Ala Lys Arg Th - #r Lys Pro Asn Gly His      545                 5 - #50                 5 - #55                 5 -      #60                                                                              - - Ile Ala Asn Arg Leu Glu Val Asp Ser Asn Th - #r Ser Ser Gln Ser        Ser                                                                                             565  - #               570  - #               575             - - Asn Ser Glu Ser Glu Thr Glu Asp Glu Arg Va - #l Gly Glu Asp Thr Pro                  580      - #           585      - #           590                  - - Phe Leu Gly Ile Gln Asn Pro Leu Ala Ala Se - #r Leu Glu Ala Thr Pro              595          - #       600          - #       605                      - - Ala Phe Arg Leu Ala Asp Ser Arg Thr Asn Pr - #o Ala Gly Arg Phe Ser          610              - #   615              - #   620                          - - Thr Gln Glu Glu Ile Gln Ala Arg Leu Ser Se - #r Val Ile Ala Asn Gln      625                 6 - #30                 6 - #35                 6 -      #40                                                                              - - Asp Pro Ile Ala Val                                                                      645                                                            - -  - - <210> SEQ ID NO 94                                                  <211> LENGTH: 56                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 94                                                        - - Gly Thr Ser His Leu Val Lys Cys Gly Trp As - #p Arg Glu Gly Phe        Cys                                                                              1               5  - #                10  - #                15              - - Val Asn Gly Gly Glu Cys Phe Met Val Lys As - #p Leu Ser Asn Pro Ser                  20      - #            25      - #            30                   - - Arg Tyr Leu Cys Lys Cys Pro Asn Glu Phe Th - #r Gly Asp Arg Cys Gln              35          - #        40          - #        45                       - - Asn Tyr Val Ile Ala Ser Phe Tyr                                              50              - #    55                                                  - -  - - <210> SEQ ID NO 95                                                  <211> LENGTH: 56                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 95                                                        - - Gly Thr Ser His Leu Val Lys Cys Asp Lys Se - #r Arg Glu Gly Phe Cys       1               5  - #                10  - #                15               - - Val Asn Gly Gly Glu Cys Phe Met Val Lys As - #p Leu Ser Asn Pro Ser                  20      - #            25      - #            30                   - - Arg Tyr Leu Cys Lys Cys Pro Asn Glu Phe Th - #r Gly Asp Arg Cys Gln              35          - #        40          - #        45                       - - Asn Tyr Val Ile Ala Ser Phe Tyr                                              50              - #    55                                                  - -  - - <210> SEQ ID NO 96                                                  <211> LENGTH: 56                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 96                                                        - - Gly Thr Ser His Leu Val Lys Cys Ala Glu Ly - #s Glu Lys Thr Phe Cys       1               5  - #                10  - #                15               - - Val Asn Gly Gly Glu Cys Tyr Lys Val Arg Il - #e Leu Ser Asn Pro Ser                  20      - #            25      - #            30                   - - Arg Tyr Leu Cys Lys Cys Pro Asn Glu Phe Th - #r Gly Asp Arg Cys Gln              35          - #        40          - #        45                       - - Asn Tyr Val Ile Ala Ser Phe Tyr                                              50              - #    55                                                  - -  - - <210> SEQ ID NO 97                                                  <211> LENGTH: 56                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 97                                                        - - Gly Thr Ser His Leu Val Lys Cys Ala Glu Ly - #s Glu Lys Thr Phe Cys       1               5  - #                10  - #                15               - - Val Asn Gly Gly Glu Cys Phe Met Val Lys As - #p Leu Ser Asn Tyr Gly                  20      - #            25      - #            30                   - - Tyr Leu Met Cys Lys Cys Pro Asn Glu Phe Th - #r Gly Asp Arg Cys Gln              35          - #        40          - #        45                       - - Asn Tyr Val Met Ala Ser Phe Tyr                                              50              - #    55                                                  - -  - - <210> SEQ ID NO 98                                                  <211> LENGTH: 56                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 98                                                        - - Gly Thr Ser His Leu Val Lys Cys Ala Glu Ly - #s Glu Lys Thr Phe Cys       1               5  - #                10  - #                15               - - Val Asn Gly Gly Glu Cys Phe Met Val Lys As - #p Leu Ser Asn Tyr Arg                  20      - #            25      - #            30                   - - Tyr Arg Met Cys Lys Cys Pro Asn Glu Phe Th - #r Gly Asp Arg Cys Gln              35          - #        40          - #        45                       - - Asn Tyr Val Ile Ala Ser Phe Tyr                                              50              - #    55                                                  - -  - - <210> SEQ ID NO 99                                                  <211> LENGTH: 56                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 99                                                        - - Gly Thr Ser His Leu Val Lys Cys Ala Glu Ly - #s Glu Lys Thr Phe Cys       1               5  - #                10  - #                15               - - Val Asn Gly Gly Glu Cys Phe Met Val Lys As - #p Leu Ser Asn Thr His                  20      - #            25      - #            30                   - - Tyr Arg Met Cys Lys Cys Pro Asn Glu Phe Th - #r Gly Asp Arg Cys Gln              35          - #        40          - #        45                       - - Asn Tyr Val Met Ala Ser Phe Tyr                                              50              - #    55                                                  - -  - - <210> SEQ ID NO 100                                                 <211> LENGTH: 56                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 100                                                       - - Gly Thr Ser His Leu Val Lys Cys Ala Glu Ly - #s Glu Lys Thr Phe Cys       1               5  - #                10  - #                15               - - Val Asn Gly Gly Glu Cys Phe Met Val Lys As - #p Leu Ser Asn Thr Lys                  20      - #            25      - #            30                   - - Tyr Arg Gly Cys Lys Cys Pro Asn Glu Phe Th - #r Gly Asp Arg Cys Gln              35          - #        40          - #        45                       - - Asn Tyr Val Met Ala Ser Phe Tyr                                              50              - #    55                                                  - -  - - <210> SEQ ID NO 101                                                 <211> LENGTH: 56                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 101                                                       - - Gly Thr Ser His Leu Val Lys Cys Ala Glu Ly - #s Glu Lys Thr Phe Cys       1               5  - #                10  - #                15               - - Val Asn Gly Gly Glu Cys Phe Met Val Lys As - #p Leu Ser Asn Pro Ser                  20      - #            25      - #            30                   - - Arg Tyr Leu Cys Lys Cys Pro Asn Glu Phe Th - #r Gly Asp Arg Cys Gln              35          - #        40          - #        45                       - - Trp Tyr Val Ile Ala Ser Phe Tyr                                              50              - #    55                                                  - -  - - <210> SEQ ID NO 102                                                 <211> LENGTH: 56                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 102                                                       - - Gly Thr Ser His Leu Val Lys Cys Ala Glu Ly - #s Glu Lys Thr Phe Cys       1               5  - #                10  - #                15               - - Val Asn Gly Gly Glu Cys Phe Met Val Lys As - #p Leu Ser Asn Pro Ser                  20      - #            25      - #            30                   - - Arg Tyr Leu Cys Lys Cys Pro Asn Glu Phe Th - #r Gly Asp Arg Cys Gln              35          - #        40          - #        45                       - - His Tyr Val Ile Ala Ser Phe Tyr                                              50              - #    55                                                  - -  - - <210> SEQ ID NO 103                                                 <211> LENGTH: 56                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 103                                                       - - Gly Thr Trp Glu Leu Val Pro Cys Gly Trp As - #p Arg Glu Gly Phe Cys       1               5  - #                10  - #                15               - - Val Asn Gly Gly Glu Cys Phe Met Val Lys As - #p Leu Ser Asn Pro Ser                  20      - #            25      - #            30                   - - Arg Tyr Leu Cys Lys Cys Pro Asn Glu Phe Th - #r Gly Asp Arg Cys Gln              35          - #        40          - #        45                       - - Asn Tyr Val Ile Ala Ser Phe Tyr                                              50              - #    55                                                  - -  - - <210> SEQ ID NO 104                                                 <211> LENGTH: 56                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 104                                                       - - Gly Thr Ser His Leu Val Lys Cys Ala Glu Ly - #s Glu Lys Thr Phe Cys       1               5  - #                10  - #                15               - - Val Asn Gly Gly Glu Cys Phe Met Val Lys As - #p Leu Ser Asn Tyr Gly                  20      - #            25      - #            30                   - - Tyr Leu Met Cys Lys Cys Pro Asn Glu Phe Th - #r Gly Asp Arg Cys Gln              35          - #        40          - #        45                       - - Asn Tyr Val Ile Ala Ser Phe Tyr                                              50              - #    55                                                  - -  - - <210> SEQ ID NO 105                                                 <211> LENGTH: 56                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 105                                                       - - Gly Thr Ser His Leu Val Lys Cys Gly Glu Gl - #u Arg Glu Gly Phe Cys       1               5  - #                10  - #                15               - - Val Asn Gly Gly Glu Cys Tyr Arg Val Lys Th - #r Leu Ser Asn Pro Ser                  20      - #            25      - #            30                   - - Arg Tyr Leu Cys Lys Cys Pro Asn Glu Phe Th - #r Gly Asp Arg Cys Gln              35          - #        40          - #        45                       - - Asn Tyr Val Met Ala Ser Phe Tyr                                              50              - #    55                                                  - -  - - <210> SEQ ID NO 106                                                 <211> LENGTH: 56                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 106                                                       - - Gly Thr Ser His Leu Val Lys Cys Gly Glu Gl - #u Arg Glu Gly Phe Cys       1               5  - #                10  - #                15               - - Val Asn Gly Gly Glu Cys Phe Met Val Lys As - #p Leu Ser Asn Tyr Gly                  20      - #            25      - #            30                   - - Tyr Leu Met Cys Lys Cys Pro Asn Glu Phe Th - #r Gly Asp Arg Cys Gln              35          - #        40          - #        45                       - - Asn Tyr Val Met Ala Ser Phe Tyr                                              50              - #    55                                                  - -  - - <210> SEQ ID NO 107                                                 <211> LENGTH: 56                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 107                                                       - - Gly Thr Ser His Leu Val Lys Cys Gly Glu Gl - #u Arg Glu Gly Phe Cys       1               5  - #                10  - #                15               - - Val Asn Gly Gly Glu Cys Tyr Arg Val Lys Th - #r Leu Ser Asn Tyr Gly                  20      - #            25      - #            30                   - - Tyr Leu Met Cys Lys Cys Pro Asn Glu Phe Th - #r Gly Asp Arg Cys Gln              35          - #        40          - #        45                       - - Asn Tyr Val Met Ala Ser Phe Tyr                                              50              - #    55                                                  - -  - - <210> SEQ ID NO 108                                                 <211> LENGTH: 56                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 108                                                       - - Gly Thr Ser His Leu Val Lys Cys Gly Glu Gl - #u Arg Glu Gly Phe Cys       1               5  - #                10  - #                15               - - Val Asn Gly Gly Glu Cys Phe Met Val Lys As - #p Leu Ser Asn Pro Ser                  20      - #            25      - #            30                   - - Arg Tyr Leu Cys Lys Cys Pro Asn Glu Phe Th - #r Gly Asp Arg Cys Gln              35          - #        40          - #        45                       - - Asn Tyr Val Ile Ala Ser Phe Tyr                                              50              - #    55                                                  - -  - - <210> SEQ ID NO 109                                                 <211> LENGTH: 56                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 109                                                       - - Gly Thr Ser His Leu Val Lys Cys Ala Glu Ly - #s Glu Lys Thr Phe Cys       1               5  - #                10  - #                15               - - Val Asn Gly Gly Glu Cys Tyr Arg Val Lys Th - #r Leu Ser Asn Tyr Gly                  20      - #            25      - #            30                   - - Tyr Leu Met Cys Lys Cys Pro Asn Glu Phe Th - #r Gly Asp Arg Cys Gln              35          - #        40          - #        45                       - - Asn Tyr Val Met Ala Ser Phe Tyr                                              50              - #    55                                                  - -  - - <210> SEQ ID NO 110                                                 <211> LENGTH: 56                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 110                                                       - - Gly Thr Ser His Leu Val Lys Cys Ala Glu Ly - #s Glu Lys Thr Phe Cys       1               5  - #                10  - #                15               - - Val Asn Gly Gly Glu Cys Tyr Arg Val Lys Th - #r Leu Ser Asn Tyr Gly                  20      - #            25      - #            30                   - - Tyr Leu Met Cys Lys Cys Pro Asn Glu Phe Th - #r Gly Asp Arg Cys Gln              35          - #        40          - #        45                       - - Asn Tyr Val Ile Ala Ser Phe Tyr                                              50              - #    55                                                  - -  - - <210> SEQ ID NO 111                                                 <211> LENGTH: 56                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 111                                                       - - Gly Thr Ser His Leu Val Lys Cys Ala Glu Ly - #s Glu Lys Thr Phe Cys       1               5  - #                10  - #                15               - - Val Asn Gly Gly Glu Cys Tyr Arg Val Lys Th - #r Leu Ser Asn Pro Ser                  20      - #            25      - #            30                   - - Arg Tyr Leu Cys Lys Cys Pro Asn Glu Phe Th - #r Gly Asp Arg Cys Gln              35          - #        40          - #        45                       - - Asn Tyr Val Ile Ala Ser Phe Tyr                                              50              - #    55                                                  - -  - - <210> SEQ ID NO 112                                                 <211> LENGTH: 56                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 112                                                       - - Gly Thr Ser His Leu Val Lys Cys Gly Glu Gl - #u Arg Glu Gly Phe Cys       1               5  - #                10  - #                15               - - Val Asn Gly Gly Glu Cys Tyr Arg Val Lys Th - #r Leu Ser Asn Pro Ser                  20      - #            25      - #            30                   - - Arg Tyr Leu Cys Lys Cys Pro Asn Glu Phe Th - #r Gly Asp Arg Cys Gln              35          - #        40          - #        45                       - - Asn Tyr Val Ile Ala Ser Phe Tyr                                              50              - #    55                                                  - -  - - <210> SEQ ID NO 113                                                 <211> LENGTH: 56                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 113                                                       - - Gly Thr Ser His Leu Val Lys Cys Gly Glu Gl - #u Arg Glu Gly Phe Cys       1               5  - #                10  - #                15               - - Val Asn Gly Gly Glu Cys Phe Met Val Lys As - #p Leu Ser Asn Tyr Gly                  20      - #            25      - #            30                   - - Tyr Leu Met Cys Lys Cys Pro Asn Glu Phe Th - #r Gly Asp Arg Cys Gln              35          - #        40          - #        45                       - - Asn Tyr Val Ile Ala Ser Phe Tyr                                              50              - #    55                                                  - -  - - <210> SEQ ID NO 114                                                 <211> LENGTH: 56                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 114                                                       - - Gly Thr Ser His Leu Val Lys Cys Gly Glu Gl - #u Arg Glu Gly Phe Cys       1               5  - #                10  - #                15               - - Val Asn Gly Gly Glu Cys Tyr Arg Val Lys Th - #r Leu Ser Asn Tyr Gly                  20      - #            25      - #            30                   - - Tyr Leu Met Cys Lys Cys Pro Asn Glu Phe Th - #r Gly Asp Arg Cys Gln              35          - #        40          - #        45                       - - Asn Tyr Val Ile Ala Ser Phe Tyr                                              50              - #    55                                                  - -  - - <210> SEQ ID NO 115                                                 <211> LENGTH: 56                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 115                                                       - - Gly Thr Ser His Leu Val Lys Cys Ala Glu Ly - #s Glu Lys Thr Phe Cys       1               5  - #                10  - #                15               - - Val Asn Gly Gly Glu Cys Tyr Arg Val Lys Th - #r Leu Ser Asn Tyr Gly                  20      - #            25      - #            30                   - - Tyr Leu Met Cys Lys Cys Pro Asn Glu Phe Th - #r Gly Asp Arg Cys Gln              35          - #        40          - #        45                       - - His Tyr Val Ile Ala Ser Phe Tyr                                              50              - #    55                                                  - -  - - <210> SEQ ID NO 116                                                 <211> LENGTH: 56                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 116                                                       - - Gly Thr Ser His Leu Val Lys Cys Gly Glu Gl - #u Arg Glu Gly Phe Cys       1               5  - #                10  - #                15               - - Val Asn Gly Gly Glu Cys Tyr Arg Val Lys Th - #r Leu Ser Asn Tyr Gly                  20      - #            25      - #            30                   - - Tyr Leu Met Cys Lys Cys Pro Asn Glu Phe Th - #r Gly Asp Arg Cys Gln              35          - #        40          - #        45                       - - His Tyr Val Ile Ala Ser Phe Tyr                                              50              - #    55                                                __________________________________________________________________________

What is claimed is:
 1. A variant of heregulin, said variant having anamino acid sequence not found in nature and the ability to bind an ErbBreceptor, wherein said variant comprises a different amino acid than insaid heregulin wherein:at residue number 177 said different amino acidis A, F, W, or Y; at residue number 178 said different amino acid is A,D, E, G, L, N, P, Q, R, T V, or W; at residue number 179 said differentamino acid is A, G, L, M, P, S or V; at residue number 180 saiddifferent amino acid is A, D, E, G, H, I, K, M, N, P, Q, or R; atresidue number 181 said different amino acid is A, G, I, L, P, or V; atresidue number 183 said different amino acid is A, G, I, L, M, S, or T;at residue number 184 said different amino acid is A, F, G, H, I, K, L,M, N, P, Q, R, S, V, or W; at residue number 185 said different aminoacid is A, G, H, I, K, L, M, N, P, Q, S, T, or V; at residue number 186said different amino acid is A, K, R, or S; at residue number 187 saiddifferent amino acid is A, E, G, I, L, M, N, P, Q, S, or T; at residuenumber 188 said different amino acid is A, H, K, N, or R; at residuenumber 195 said different amino acid is A, H, N, Q, R, or S; at residuenumber 197 said different amino acid is A, F, L, V, or W; at residuenumber 198 said different amino acid is A, H, K, R, or S; at residuenumber 200 said different amino acid is A, H, R, or S; at residue number201 said different amino acid is G, H, I, L, M, P, R, S, T, or V; atresidue number 205 said different amino acid is A, F, H, I, K, R, T, V,W, or Y; at residue number 206 said different amino acid is A, F, G, H,I, K, L, M, P, R, S, V, W, or Y; at residue number 207 said differentamino acid is F, H, I, L, P, R, V, W, or Y; at residue number 208 saiddifferent amino acid is A, H, K, R, or S; at residue number 209 saiddifferent amino acid is G, M, P, S, T, or V; at residue number 211 saiddifferent amino acid is A, H, R, or S; at residue number 213 saiddifferent amino acid is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,T, W, or Y; at residue number 214 said different amino acid is A, C, D,E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W, or Y; at residue number 215said different amino acid is A, C, D, F, H, I, K, L, M, N, P, Q, R, S,T, V, W, or Y; at residue number 216 said different amino acid is A, G,L, M, P, or V; at residue number 223 said different amino acid is A, F,H, R, S, or W; or at residue number 226 said different amino acid is A,G, L, or P; wherein said residue numbers correspond to residue numbersof native human heregulin-β1 (SEQ ID NO: 93) numbered from theN-terminus; and wherein said heregulin variant comprises a portion thatis at least 70% identical to the portion from about residue 175 to aboutresidue 230 of native human heregulin-β1 (SEQ ID NO: 93).
 2. Theheregulin variant of claim 1 wherein:at residue number 178 saiddifferent amino acid is A, D, E, G, L, N, P, Q, R, T V, or W; at residuenumber 179 said different amino acid is A, G, L, M, P, S or V; atresidue number 187 said different amino acid is A, E, G, I, L, M, N, P,Q, S, or T; at residue number 195 said different amino acid is A, H, N,Q, R, or S; at residue number 207 said different amino acid is F, H, I,L, P, R, V, W, or Y; at residue number 211 said different amino acid isA, H, R, or S.
 3. The heregulin variant of claim 1 wherein saidheregulin is a human heregulin.
 4. The heregulin variant of claim 3wherein said human heregulin is heregulin-β1.
 5. The heregulin variantof claim 1 wherein said heregulin variant is a fragment.
 6. Theheregulin variant of claim 5 wherein said fragment comprises residuescorresponding to a portion of human heregulin-β1 extending from aboutresidue 175 to about residue
 230. 7. The heregulin variant of claim 6wherein said heregulin is a human heregulin.
 8. The heregulin variant ofclaim 7 wherein said human heregulin is human heregulin-β1.
 9. A variantof human heregulin-β1, said variant having an amino acid sequence notfound in nature and the ability to bind an ErbB receptor, wherein saidvariant comprises an amino acid substitution selected from the groupconsisting of;S177W; H117E, R, or A; V180Q, I, or E; K181P or A; A183G;E184V, W, R, G, or N; K185S, Q, or G; E186R; K187E or A; E195Q; M198R orK; D201T or I; P205T or Y; S206K, H, G, P, or R; R207Y; Y208R or L;L209M or G; K211R; P213S, T, N, or K; N214L, K, S, or E; F216M; andN223H or W; wherein said residue numbers correspond to residue numbersof native human heregulin-β1 (SEQ ID NO: 93) numbered from theN-terminus; and wherein said heregulin variant comprises a portion thatis at least 70% identical to the portion from about residue 175 to aboutresidue 230 of native human heregulin-β1 (SEQ ID NO: 93).
 10. Theheregulin variant of claim 9 wherein said heregulin variant comprises aset of aminio acid substitutions selected fromA183G, E184W, K185D,E186R, K187E, T188G, M226I; A183D, E184K, K185S, E186R, K187E, T188G,M226I; F197Y, M198K, K200R, D201I, M226I; P205Y, S206G, R207Y, Y208L,L209M; P205Y, S206R, R207Y, Y208R, L209M, M226I; P205T, S206H, R207Y,Y208R, L209M; P205T, S206K, R207Y, Y208R, L209G; N223W, M226I; N223H,M226I; S177W, H178E, K181P, A183G, E184W, K185D, E186R, K187E, T188G,M226I; P205Y, S206G, R207Y, Y208L, L209M, M226I; A183G, K185E, E186R,K187E, T188G, F197Y, M198R, D201T; A183G, K185E, E186R, K187E, T188G,P205Y, S206G, R207Y, Y208L, L209M; A183G, K185E, E186R, K187E, T188G,F197Y, M198R, D201T, P205Y, S206G, R207Y, Y208L, L209M; A183G, K185E,E186R, K187E, T188G, M226I; F197Y, M198R, D201T, P205Y, S206G, R207Y,Y208L, L209M; F197Y, M198R, D201T, P205Y, S206G, R207Y, Y208L, L209M,M226I; F197Y, M198R, D201T, M226I; A183G, K185E, E186R, K187E, T188G,F197Y, M198R, D201T, M226I; A183G, K185E, E186R, K187E, T188G, P205Y,S206G, R207Y, Y208L, L209M, M226I; A183G, K185E, E186R, K187E, T188G,F197Y, M198R, D201T, P205Y, S206G, R207Y, Y208L, L209M, M226I; F197Y,M198R, D201T, P205Y, S206G, R207Y, Y208L, L209M, N223H, M226I; andA183G, K185E, E186R, K187E, T188G, F197Y, M198R, D201T, P205Y, S206G,R207Y, Y208L, L209M, N223H, M226I.
 11. The heregulin variant of claim 10wherein said heregulin variant comprises a set of amino acidsubstitutions selected fromA183G, E184W, K185D, E186R, K187E, T188G,M226I; P205Y, S206G, R207Y, Y208L, L209M; N223H, M226I; P205Y, S206G,R207Y, Y208L, L209M, M226I; A183G, K185F, E186R, K187E, T188G, F197Y,M198R, D201T; A183G, K185E, E186R, K187E, T188G, M226I; F197Y, M198R,D201T, P205Y, S206G, R207Y, Y208L, L209M; F197Y, M198R, D201T, P205Y,S206G, R207Y, Y208L, L209M, M226I; F197Y, M198R, D201T, M226I; A183G,K185E, E186R, K187E, T188G, F197Y, M198R, D201T, M226I; A183G, K185E,E186R, K187E, T188G, P205Y, S206G, R207Y, Y208L, L209M, M226I; A183G,K185E, E186R, K187E, T188G, F197Y, M198R, D201T, P205Y, S206G, R207Y,Y208L, L209M, M226I; F197Y, M198R, D201T, P205Y, S206G, R207Y, Y208L,L209M, N223H, M226I; and A183G, K185E, E186R, K187E, T188G, F197Y,M198R, D201T, P205Y, S206G, R207Y, Y208L, L209M, N223H, M226I.
 12. Theheregulin variant of claim 9 wherein said heregulin variant is afragment.
 13. The heregulin variant of claim 12 wherein said fragmentcomprises residues corresponding to a portion of human heregulin-β1extending from about residue 175 to about residue
 230. 14. A nucleicacid molecule encoding the heregulin variant of claim
 1. 15. A vectorcomprising the nucleic acid molecule of claim
 14. 16. A host cellcomprising the vector of claim
 15. 17. A method of producing a variantof a heregulin comprising:(a) culturing the host cell of claim 16 underconditions that allow expression of the heregulin variant; and (b)recovering the heregulin variant from the culture.
 18. A method ofproducing a heregulin variant comprising modifying the heregulin variantof claim 1 to produce a modified heregulin variant, wherein the modifiedheregulin variant retains the ability to bind an ErbB receptor.
 19. Themethod of claim 18 wherein said modifying step comprises introducing amodification selected from the group consisting of an amino acidsubstitution, an insertion of at least one amino acid, a deletion of atleast one amino acid, and a chemical modification.
 20. A compositioncomprising the heregulin variant of claim 1 and a pharmaceuticallyacceptable carrier.
 21. The heregulin variant of claim 1 wherein:atresidue number 178 said different amino acid is A, D, E, G, L, N, P, Q,R, T, V, or W; at residue number 179 said different amino acid is A, G,L, M, P, S or V; or at residue number 207 said different amino acid isF, H, I, L, P, R, V, W, or Y; and wherein said heregulin variant has agreater specificity for the ErbB-4 receptor, relative to the ErbB-3receptor, than the heregulin from which said heregulin variant isderived.
 22. The heregulin variant of claim 21 wherein said heregulin isa human heregulin.
 23. The heregulin variant of claim 22 wherein saidhuman heregulin is heregulin-β1.
 24. The heregulin variant of claim 23wherein said amino acid substitution is selected from H178E and R207P.25. The heregulin variant of claim 21 wherein said heregulin variant isa fragment.
 26. The heregulin variant of claim 25 wherein said fragmentcomprises residues corresponding to a portion of human heregulin-β1extending from about residue 175 to about residue
 245. 27. A heregulinvariant having an amino acid sequence not found in nature and theability to bind an ErbB receptor,wherein said variant comprises amethionine residue in place of amino acid residues corresponding toresidue numbers 228 to 231 of native human heregulin-β1 (SEQ ID NO: 93)numbered from the N-terminus and said heregulin variant comprises aportion that is at least 70% identical to the portion from about residue175 to about residue 230 of native human heregulin-β1 (SEQ ID NO: 93),said heregulin variant having a greater specificity for the ErbB-4receptor, relative to the ErbB-3 receptor, than a heregulin that differsfrom the heregulin variant only in that the heregulin comprises saidamino acid residues corresponding to residue numbers 228 to 231 in placeof said methionine.
 28. The heregulin variant of claim 27 additionallycomprising the amino acid substitution H178L.
 29. The heregulin variantof claim 27 wherein said heregulin is a human heregulin.
 30. Theheregulin variant of claim 29 wherein said human heregulin isheregulin-β1.
 31. The heregulin variant of claim 27 wherein saidheregulin variant is a fragment.
 32. The heregulin variant of claim 30wherein said fragment comprises residues corresponding to a portion ofhuman heregulin-β1 extending from about residue 175 to about residue245.